Methods of expanding myeloid cell populations and uses thereof

ABSTRACT

The present disclosure relates to a method of expanding myeloid progenitor cells by culturing an initial population of cells in a medium comprising a mixture of cytokines and growth factors that promote growth and expansion of the myeloid progenitor cells. The expanded cell population provides a source of cells as therapeutic treatments for neutropenia and/or thrombocytopenia arising in patients subjected to myeloablative therapy and hematopoietic stem cell transplantation.

1. CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/481,715, filed Sep. 9, 2014, which is a divisional of U.S.patent application Ser. No. 13/540,471, filed Jul. 2, 2012, which is adivisional of U.S. patent application Ser. No. 11/259,592, now U.S. Pat.No. 8,252,587, filed Oct. 25, 2005, which claims benefit under 35 U.S.C.§ 119(e) to application Ser. No. 60/622,318, entitled “Methods ofExpanding Myeloid Cell populations and Uses Thereof”, filed Oct. 25,2004, the disclosure of each is incorporated herein by reference in itsentirety.

2. TECHNICAL FIELD

The present disclosure relates to compositions and methods forshort-term reconstitution of the hematopoietic system, and uses thereoffor treatment of conditions associated with impaired or ablatedhematopoiesis.

3. INTRODUCTION

Hematopoietic stem cell transplantation (HSCT) is a standard therapy forvarious hematological malignancies, and in some cases, is the onlyviable treatment option, particularly when the disease is refractory tochemotherapy. Bone marrow, peripheral blood, or cord blood serve astypical sources of hematopoietic stem cells (HSCs), but cells fromperipheral blood display more rapid engraftment characteristics and maybe mobilized by treatment of the donor with cytokines G-CSF, GM-CSF, orwith cytoreductive drugs. Cord blood is readily available, shows lowerincidences of graft versus host disease but is characterized by delayedengraftment. Prior to transplantation, the recipient is givenmyeloablative doses of chemotherapy and/or radiation to treat theunderlying disease and make the recipient suitable for engraftment ofthe donor HSCs.

Generally, there are two types of HSCT: autologous and allogeneic.Autologous transplantation involves infusion of a recipient's own cellsfollowing myeloablative treatment. Autologous cell transplants minimizethe risk of graft versus host disease (GVHD) and result in reducedcomplications. Since chemotherapy with myeloablative agents is used toeliminate malignant cells in the HSC preparation, autologoustransplantation is problematic if the disease is unresponsive tochemotherapy. Allogeneic transplantation involves infusion of donor stemcells, typically using a donor that matches the recipient's MHC. Anadvantage of allogeneic transplants is the accompanying cell-mediatedgraft versus host reaction that may develop against malignant cells.However, matched unrelated donor (MUD) transplants are also associatedwith a stronger graft versus host reaction, and thus result in highermortality rates.

There are several added complications associated with myeloablativetherapy and HSCT, most notably neutropenia and thrombocytopenia. Bothconditions arise from impaired hematopoiesis and the inability of thehematopoietic system to adequately replenish the terminallydifferentiated myeloid cell associated with each disorder. Neutropeniaand thromobocytopenia may also develop from other causes of impairedhematopoiesis, such as unintended exposure to lethal doses of ionizingradiation, inherited immunodeficiencies, viral infections affecting thebone marrow, and metabolic disorders (e.g., vitamin deficiencies).

Neutropenia is a condition characterized by abnormally low numbers ofwhite blood cells, particularly neutrophils, which are short lived andrepresent the most abundant leukocyte in the peripheral blood.Neutrophils and other polymorphonuclear leukocytes migrate to sites ofinfection through the action of various chemokines to provide a criticalimmune response against the infectious agents. During the time periodrequired for recovery of the hematopoietic system following transplant,the transplant recipient has low levels of circulating neutrophils andis susceptible to bacterial and fungal infections, particularly toopportunistic infections by commonly occurring microorganisms, such asPseudomonas aeruginosa and Aspergillus fumigatus. Prolonged neutropenia,particularly those resulting from delayed engraftment of donor HSCs,increases the probability of infection and is associated with highmortality rates.

One standard therapy for neutropenia is administration of G-CSF. Thiscytokine promotes granulocyte development and also enhances the immuneeffector responses of neutrophils. G-CSF accelerates recovery followinghematopoietic stem cell transplantation, but it may not be effective forsubjects treated with high dose chemotherapy in the absence of HSCTbecause of the low numbers of responsive hematopoietic stem cells in thepatient.

Another therapeutic approach involves infusion of neutrophils(granulocyte transfusions) as a temporary measure to protect againstinfections. Following treatment of the donor with G-CSF or GM-CSF, cellsare collected by leukapheresis of peripheral blood and administered intothe recipient to elevate circulating neutrophil levels (see, e.g., Lin,Y-W et al., J. Clin. Microbiol. 41(10):4892-4893 (2003)). Subjectstreated by this method show increased survival, but the clinicalefficacy of this approach appears uncertain, possibly owing to the shortlife span of differentiated neutrophils following transplant or due toadverse effects of storage on neutrophil activity (McCullough, J. etal., Transfusion 23(1):20 (1983)). Moreover, because the efficacy ofneutrophil transfusion correlates with the number of cells administered,the limited availability of donor cells, usually from matched sibling orhaplomatched parent, and the inability to store the cells may limit thegeneral applicability of this approach.

Thrombocytopenia is a condition associated with another terminallydifferentiated cell of the myeloid lineage, the megakaryocyte, and ischaracterized by abnormally low levels of platelets. Platelets areessential to the blood clotting process and are needed to limit leakageof erythrocytes from blood vessels. Below normal levels of plateletsresult in increased risk of bleeding, and may result in spontaneousbleeding when platelet levels drop below a critical level.Thromobocytopenia may arise from impaired platelet production and/orincreased rate of removal. Occurrence of thrombocytopenia in the HSCTsetting results from the impaired development of megakaryocytes, and isheightened by delayed engraftment of HSCs, complications frominfections, and incidences of GVHD. As with neutropenia, managingthrombocytopenia is critical after any myeloablative treatment tominimize life-threatening complications.

Platelet transfusion is a routine therapy to treat thromobocytopenia,and is effective in reducing serious bleeding problems associated withlow platelet levels. Use of platelets from MHC-matched donors minimizesany adverse immune response against the donor platelets. The associationbetween infections and thrombocytopenia, however, suggests thatneutropenia may complicate the thrombocytopenic condition, requiringmore frequent transfusions in such patients. In addition, use of G-CSFtherapy for treating neutropenia is contraindicated for thrombocytopeniabecause of accelerated platelet destruction correlated with G-CSFadministration.

Although the infusion of immune protective cells for neutropenia andtransfusions of platelets for thromobocytopenia remain viable approachesfor treating these conditions, it is desirable to increase the durationof protection, and for neutropenia, to have available a more consistentsupply of cells for administration. Moreover, treatments capable ofaddressing both disorders concurrently, rather than separately, willimprove management of these complications of impaired hematopoiesis.

4. SUMMARY

The present disclosure describes compositions and methods useful in thetreatment of complications arising from impaired hematopoiesis. Theinability to generate terminally differentiated cells of the myeloidlineage in subjects with insufficient hematopoiesis leads to a number oflife threatening conditions, most notably neutropenia andthrombocytopenia. By transiently replacing the cell populationassociated with these conditions, complications arising from depletionof the differentiated myeloid cells are ameliorated or prevented untilsuch time as the patient's endogenous or reconstituted hematopoieticsystem can regenerate.

Accordingly, in one aspect, the present invention provides methods forpreparing therapeutic compositions of myeloid progenitor cells forreconstituting hematopoiesis in a mammalian host, comprising: a)culturing ex vivo a starting cell population including hematopoieticstem cells in a suitable culture medium to expand said cell populationand increase the number of myeloid progenitor cells within said cellpopulation; and b) resuspending said myeloid progenitor cells in apharmaceutically acceptable medium suitable for administration to amammalian host. Advantageously, as demonstrated herein, the initialpopulation of cells may be derived from an allogeneic donor or, stillmore preferably, from a plurality of allogeneic donors, resulting in exvivo expanded allogeneic myeloid progenitor cells obtained from one or aplurality of donors. The donors may be related or unrelated to eachother, and in the transplant setting, related or unrelated to therecipient.

Surprisingly, as further demonstrated herein, the present inventors havedetermined that the expanded myeloid progenitor cells of the presentinvention can be cryopreserved after expansion and still retain theirfunctionality in reconstituting hematopoiesis in a therapeutic setting,including granulocyte/macrophage progenitors. Although hematopoieticstem cells are known to retain their functionality aftercryopreservation, see, e.g., U.S. Pat. No. 5,004,681, thecryopreservation of their more differentiated progeny has not beenuniformly successful, thus complicating their practical implementationas a clinical cell-based therapy. The expanded myeloid progenitorsprovided herein have this advantageous feature, and in a preferredembodiment are cryopreserved prior to resuspension and administration toa patient.

The initial population of cells for expansion may be derived fromperipheral blood, mobilized peripheral blood, umbilical cord blood, bonemarrow, and/or other organs known to harbor hematopoietic stem cells,such as fetal liver. Cell populations may be mixtures of cells asobtained from a source or cells isolated, particularly as an enriched orsubstantially pure population, based on a desired cell marker phenotype(e.g., CD34+ and/or CD90+ and/or AC133+ and/or ALDH+ cells). Preferably,the starting cell population is enriched for HSC based on the presenceof the cell marker CD34+ or CD90+; and still more preferably, thestarting cell population is purified HSC that are both CD34+ and CD90+,particularly for expansion of human myeloid progenitor cells. In afurther embodiment, the cells may also have the cell marker phenotypeLin^(neg/low).

In another aspect, the invention provides therapeutic compositionsincluding the expanded myeloid progenitor cells resulting from theinventive methods. In one embodiment, the therapeutic compositioncomprises or consists essentially of expanded myeloid progenitor cellsin a pharmaceutically acceptable carrier. In another embodiment, thetherapeutic composition comprises or consists essentially of expandedmyeloid progenitor cells cryopreserved in a cryopreservation medium. Ina preferred embodiment, the expanded myeloid progenitor cells areallogeneic, and still more preferably, the expanded myeloid progenitorcells are a mixture of allogeneic myeloid progenitor cells. The mixtureof allogeneic myeloid progenitor cells may comprise at least a partialmismatch at the MHC, where the MHC mismatch is between the MHC of thevarious donors or between the donors and the recipient, or a full orcomplete mismatch at the MHC. Accordingly such mixtures of allogeneiccells may comprise a partial mismatch at the MHC between some cells anda full mismatch between other cells in the population. In a particularembodiment, progenitor cells that undergo temporary engraftment (e.g.,progenitor cell populations occurring early in the myeloid lineage) areselected to have a match or a partial mismatch at the MHC while moredifferentiated progenitor cells have a partial or full mismatch at theMHC.

In a further embodiment, the mixtures of allogeneic myeloid progenitorcells are mixtures of isolated cells. These include isolated CMPs,isolated GMPs, isolated MEPs, or combinations thereof. Cells for themixtures may be obtained from unexpanded myeloid progenitor cells, orfrom ex vivo expanded cell cultures described herein. For mixtures ofallogeneic expanded cells, the allogeneic cells may be mixed prior toexpansion or subsequent to expansion.

In another aspect, the present disclosure provides methods of generatingmyeloid progenitor cells through their ex vivo expansion in culture. Inthe methods, cells capable of producing myeloid progenitor cells suchas, e.g., hematopoeitic stem cells (HSC), are contacted with a culturemedium comprising a cytokine and growth factor mixture that supportsexpansion of myeloid progenitor cells, and the cells are then culturedunder suitable conditions that facilitate their expansion. Suitablecytokines for ex vivo expansion purposes are selected from IL-1 (i.e.,IL-1β), IL-3, IL-6, IL-11, G-CSF, GM-CSF, and analogs thereof. Suitablegrowth factors for ex vivo expansion purposes are selected from c-kitligand (SCF or SF), FLT-3 ligand (FL), thrombopoietin (TPO),erythropoietin (EPO), and analogs thereof. As used herein, analogsinclude variants of the cytokines and growth factors having thecharacteristic biological activity of the naturally occurring forms.

In a preferred embodiment, the medium is a chemically-defined mediumlacking undefined (qualitatively or quantitatively) components,including cell-based expansion materials such as stromal cells or otherfeeder cells. Significantly, the inclusion of such materials can beproblematic from a manufacturing and regulatory perspective, and theselection and development of a chemically-defined alternative thatappropriately expands the desired cell types represents another yetsignificant contribution of the present invention.

In one embodiment, the cytokine and growth factor mixture in its basecomposition has stem cell factor (SCF), FLT-3 ligand (FL), andthromobopoietin (TPO). In preferred embodiments, the cytokine and growthfactor mixture has an additional cytokine selected from IL-3, IL-6,IL-11, G-CSF, GM-CSF, and combinations thereof, and particularly fromIL-3, IL-6, IL-11, and combinations thereof. Thus, in one embodiment,the cytokine and growth factor mixture has the composition SCF, FL, TPO,and IL-3 while in another embodiment, the mixture has the compositionSCF, FL, TPO, and IL-6. One combination of the additional cytokine isIL-6 and IL-11 such that the cytokine and growth factor mixture has thecomposition SCF, FL, TPO, IL-6 and IL-11.

Forms of the cytokines and growth factors are their naturally occurringproducts, recombinant products, variants or analogs, or modified formshaving similar biological activity to the naturally occurring forms. Thecytokines are chosen to be active on the cells used for expansion, andthus their source will generally reflect the origin of the initial cellsused for expansion, although this correspondence between the form of thecytokine and the origin of the cells need not be rigorous sincecross-reactivity between forms is known for various cytokines and growthfactors, and is readily testable. Thus, in one embodiment the cytokinesused are recombinant human (rhu) rhuIL-1, rhuIL-1β), rhuIL-3, rhuIL-6,rhuIL-11, rhuG-CSF, rhuGM-CSF, or analogs thereof. Similarly, the growthfactors used are recombinant human rhuSCF, rhuFL, rhuTPO, rhuEPO, oranalogs thereof.

The starting cell population is cultured under conditions that supportexpansion of myeloid progenitor cells to defined levels. Expandedmyeloid progenitor cells obtained in accordance with the presentinvention generally comprise common myeloid progenitor cells (CMP),granulocyte/macrophage progenitor cells (GMP), andmegakaryocyte/erythroid progenitor cells (MEP). Thus, in one aspect, theex vivo expanded cultures comprise expanded CMPs in which the CMP cellpopulation is expanded at least about 5 fold, about 10 fold, about 20fold, or at least about 30 fold. In the final cell culture preparation(e.g., at the time of cell harvest), the expanded culture comprises aCMP population which is at least about 0.5%, at least about 1%, at leastabout 2%, at least about 5%, and at least about 10% of the total cellsin the culture.

In another aspect, the ex vivo expanded cultures comprise expanded GMPsin which the GMP cell population is expanded at least about 10 fold,about 20 fold, about 40 fold, and preferably at least about 80 fold. Inthe final cell culture preparation, the expanded cultures comprise a GMPpopulation which is at least about 10%, at least about 20%, at leastabout 30%, and preferably at least about 50% of the total cells inculture. The MEP cell population in the cultures is expanded at leastabout 0.1 fold, about 2 fold, about 5 fold, and at least about 10 fold.In the final cell culture preparation, the expanded cultures comprise aMEP cell population which is at least about 0.5%, about 1%, about 2%,and at least about 5% of the total cells in the culture.

Collectively, the combined total of the myeloid progenitor cells in thefinal cell culture preparation is at least about 25%, at least about40%, more preferably greater than about 50%, still more preferably atleast about 60% or 70%, most preferably at least about 80% or 90%, andideally at least about 95% of the total cells in the expanded culture.

Cells prepared by ex vivo expansion may be resuspended in apharmaceutically acceptable carrier and used directly or alternativelymay be subjected to processing by various cell purification techniquesavailable to the skilled artisan, such as FACS sorting, magneticaffinity separation, and immunoaffinity columns. Cell populationsisolated from the expanded cultures include, among others, isolatedmyeloid progenitor cells, isolated CMP, isolated GMP and isolated MEP.Preferably the isolated cell population is a substantially purepopulation of cells.

The cells described herein have various applications in therapeutic andnon-therapeutic settings. In therapeutic applications, the cells areused to treat subjects with impaired or ablated hematopoiesis. Cells areadministered into a subject, such as by intravenous infusion, in asufficient amount to provide a therapeutic benefit, eitherprophylatically to lessen the occurrence of adverse conditionsassociated with impaired hematopoiesis or to treat a subject alreadysuffering from complications associated with impaired hematopoiesis. Ina further aspect, the cells are used to treat subjects in the HSCTsetting, either concurrently with or subsequent to the HSCT.

Neutropenia and thrombocytopenia are associated with impairedhematopoiesis, particularly where the subject has undergonemyeloablative therapy, although the conditions can occur in othercontexts. The cells described herein are applicable for the treatment ofthese conditions, either as a prophylactic measure to reduce theoccurrence of the conditions or when the subject is afflicted with theconditions. As with the other therapeutic applications, cells intherapeutic form include expanded or unexpanded myeloid progenitorcells. Since neutropenia and thrombocytopenia are associated withinsufficiency of specific myeloid cell types, the cell populationschosen can be tailored to the specific condition being treated. In oneembodiment, the cells are CMPs, which is useful for either conditionsince CMPs ultimately develop into neutrophils and megakaryocytes. Insituations of neutropenia, the cells can be GMPs since GMPs ultimatelydevelop into neutrophils. In situations for thrombocytopenia, the cellscan be MEPs since MEPs ultimately develop into megarkaryocytes. As willbe apparent to the skilled artisan, combinations of cell populationsfind application in treating these conditions, as well as the conditionsdescribed above. Combinations of isolated GMP and MEP are useful fortreating neutropenia and thrombocytopenia concurrently. Addition of CMPsto the combination should provide more prolonged protection arising fromthe temporary engraftment of CMPs and subsequent production ofneutrophils and megakaryocytes. Other combinations include CMP and GMPif neutropenia is the main focus of treatment, while the combination maybe CMP and MEP if thrombocytopenia is the main focus of treatment.

Cells are administered by methods well known in the art. In oneembodiment, the administration is by intravenous infusion. Theadministration of cells can be through a single administration orsuccessive administrations. When successive administrations areinvolved, different cells numbers and/or different cells populations maybe used for each administration. Thus in one embodiment, a firstadministration is of a cell population or a combination of cellpopulations that provide an immediate therapeutic benefit as well asmore prolonged effect (CMP+GMP+neutrophils) while the secondadministration includes cells providing prolonged effect (e.g., CMP) toextend the therapeutic effect of the first administration. These andother strategies will be apparent to the skilled artisan.

In further embodiments, the myeloid progenitor cells described hereinare used in combination with other therapeutic compounds that areeffective in treating the conditions associated with impairedhematopoiesis, and/or complications of neutropenia and thrombocytopenia.In one embodiment, the adjunctive treatments are antibacterial,antifungal, or antiviral compounds for preventing opportunisticinfections or infections already in progress in the subject.

In another embodiment, the adjunctive treatments are therapeuticcompounds that augment the differentiation of myeloid progenitor cellsin the myeloid pathway. These adjunctive treatments have the effect ofinducing differentiation and mobilization of myeloid progenitor cellsthat are endogenous, or administered to the subject as part of thetherapy. In one embodiment, particularly for treating or preventingneutropenia, G-CSF or GM-CSF is administered concurrently with orsubsequent to cell administration. Another adjunctive treatment involvesadministration of EPO or TPO, in particular as adjunctive treatments forthrombocytopenia since EPO induces differentation of MEK intoproerythroblasts and mature erythroid cells while TPO appears to inducegrowth and differentiation of hematopoietic stem cells and early myeloidprogenitor cells into megakaryocytes and mature platelets.

In a final aspect, the disclosure provides kits containing the cytokinesand growth factor mixture, initial cells for expansion, media and othernecessary components for carrying out the ex vivo expansion methods.Kits directed to use of the cell populations, expanded or unexpanded,for therapeutic applications, such as for treatments for neutropenia andthrombocytopenia, are provided. The kits may further include, by way ofexample and not limitation, buffers, labels, reagents, and instructionsfor methods of using the kits.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C is a schematic of an experimental design showing (FIG. 1A)Cell populations sorted and analyzed. (B) (FIG. 1B) Derivation ofmyeloid progenitors from HSC in culture, culture derived MP cells can beused fresh or cryopreserved. (C) (FIG. 1C) The use of myeloidprogenitors to protect neutropenic mice from a fungal challenge.

FIG. 2A-2B shows derivation of myeloid progenitors from HSC in culture.

FIG. 3 shows protection of neutropenic mice by allogeneicculture-derived myeloid progenitors.

FIG. 4A-4B shows a comparison of fresh (FIG. 4A) and cryopreserved (FIG.4B) myeloid progenitors.

FIG. 5A-5C shows reconstitution with myeloid progenitors in mice.

FIG. 6A-6B shows dose response of protection by culture-derived myeloidprogenitors.

FIG. 7 shows time to effective protection using myeloid progenitors inmice.

FIG. 8 shows protection of neutropenic mice by mixed allogeneicculture-derived myeloid progenitors.

FIG. 9A-9B shows radioprotective ability of completely MHC mismatchedallogeneic cultured progenitors (FIG. 9A) and detectable donor chimerism(FIG. 9B).

FIG. 10 shows 30 day radioprotection from lethally irradiated micetransplanted with fresh and frozen completely MHC mismatched allogeneicmyeloid progenitors.

FIGS. 11A-11B shows human expansion data in 7 ml AFC bags and flasks.

FIG. 12 shows photographs of cells from human myeloid progenitorscultures and treated with growth factors.

FIG. 13A-13B shows human expansion data in 7 ml AFC bags.

FIG. 14A-14B shows human expansion data in 72 ml AFC bags.

FIG. 15A-15B shows colony formation of human myeloid progenitors.

FIG. 16 shows FACS analysis of human myeloid progenitors cultures forstem and progenitor populations.

FIG. 17A-17B show the effect of IL-3 and IL-6, alone and in combinationon human myeloid progenitors cells.

FIG. 18A-18C shows a result of a colony formation assay of the myeloidprogenitors cultures with IL-3, IL-6 or in combination.

FIG. 19 show the absolute numbers of CFUs in myeloid progenitorscultures with IL-3, IL-6 or in combination.

FIG. 20A-20B shows responsiveness of human myeloid progenitors cellstowards G-CSF.

FIG. 21 is a schematic to show responsiveness of human myeloidprogenitors cells to G-CSF in vivo.

FIG. 22 is FACS analysis of mouse bone marrow and spleen showingengraftment of human MPc one week after transplantation and theirresponse to G-CSF.

FIG. 23 is FACS phenotype of the human culture-derived myeloidprogenitors in NOD/SCID mice.

6. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present disclosure describes methods of generating cells useful forthe treatment of conditions associated with a deficient or impairedhematopoietic system, such as the neutropenia and thrombocytopenia thatdevelops following myeloablative therapy and hematopoietic stem celltransplantation, chemotherapy for malignant diseases, or as aconsequence of unintentional exposure to high doses of ionizingradiation. The expanded cells described herein comprise committedmyeloid progenitor cells (MP) generated by contacting an initialpopulation of stem cells and progenitor cells with a defined cytokineand growth factor mixture permissive for the development of thecommitted myeloid progenitor cells. Under the defined cultureconditions, myeloid progenitor cells expand preferentially to definedlevels. The expanded cells are used as a whole, or subjected topurification to provide isolated cells having a defined cell markerphenotype and a characteristic differentiation potential. Isolated cellpopulations include common myeloid progenitors (CMP),granulocyte/macrophage progenitors (GMP), megakaryocyte/erythroidprogenitors (MEP), and combinations thereof. Infusion of these committedmyeloid progenitor cells into an immunodeficient patient results inshort term engraftment and/or production of terminally differentiatedcells of the myeloid lineage. This provides a temporary but prolongedreplenishment of terminally differentiated cells, particularlyneutrophils and megakaryocytes, thereby complementing the period ofdeficient hematopoiesis.

In the HSCT field, expansion techniques have been mainly directedtowards increasing the population of HSCs for the purposes oftransplantation and permanent restoration of hematopoiesis (Devine, S.M. et al., Bone Marrow Transplantation 31:241-252 (2003); Henschler, R.et al., Blood 84(9):2898-2903 (1994); Bhatia, M. et al., J. Exp. Med.186:619-624 (1997)). Combinations of cytokines and growth factorsemployed generally attempt to cause preferential expansion of HSCs whilelimiting their differentiation into committed cells of the myeloid andlymphoid lineages. The number of HSCs expanded in the context of HSCT isespecially relevant since the engraftment characteristics of infusedcells and survival of the transplant recipient is correlated withincreasing numbers of infused HSCs, particularly where there is amismatch at the MHC of the donor and recipient (Ketterer N. et al.,Blood 91:3148-3155 (1998)). Culture conditions that inducedifferentiation of the stem cells are undesirable because of the lowernumbers of HSCs produced. Because HSCs have self-renewing capacity, longterm cultures are used in some instances to select for self-replenishingHSC populations (Piacibillo, W. et al., Blood 93(11):3736-3749 (1999)).

In contrast, committed myeloid progenitor cells have limited or no selfrenewing capacity, and thus culture conditions suitable for HSCexpansion are not optimal for expansion of these cells. On the otherhand, the presence of cytokines that promote rapid development of thecultured cells into terminally differentiated cells (e.g., neutrophilsand megakaryocytes) is undesirable because these expanded cellpopulations may not provide the prolonged protection afforded byinfusion of less differentiated progenitor cells that are found ininitial phases of the myeloid differentiation pathway (Reichle, A. etal., Bone Marrow Transplantation 32:299-305 (2003); Zimmerman, T. M. etal., Bone Marrow Transplantation 26:505-510 (2000); Reiffers, J. et al.,Lancet 354:1092-1093 (1999)). Thus, the expansion approach describedherein limits the generation of HSCs while increasing the numbers MPs,particularly CMP and GMP cells. HSCs present in the expanded cellpopulation are principally short-term repopulating hematopoietic stemcells (ST-HSC), and are generally less than 10% of the expanded cells,more preferably less than 5%, and typically in the range of 2-5% of theexpanded cell population. The expansion methods are applicable to cellsused in the autologous or allogeneic transplant setting.

Advantageously, to further increase the number of cells available forexpansion and/or therapy and to make widespread clinical applicationcommercially feasible, the cell populations described herein preferablyinclude mixtures of allogeneic myeloid progenitor cells. Typically,allogeneic cells are not used for HSCTs because of possible GVHD andhost-versus-graft reactions, both of which can delay engraftment of HSCsin the transplant recipient. Instead, a single donor having a completeor partial match at the MHC is generally used as the source of HSCs.Unlike the HSCT context, however, the use of allogeneic committedmyeloid progenitor cells that do not match with the MHC of the host isnot adverse to their therapeutic effectiveness since permanentengraftment is neither the purpose nor the effect produced by infusionof these differentiated cells. Loss of HSCs, and long-term repopulatingHSCs in particular, also is not detrimental to the therapeutic effectsince temporary protection against neutropenia and/or thrombocytopeniais provided by the committed myeloid progenitor cells. The mixtures ofallogeneic myeloid cells may be prepared from expanded or unexpandedcells.

6.1 Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings:

“Allogeneic” refers to deriving from, originating in, or being membersof the same species, where the members are genetically related orgenetically unrelated but genetically similar. An “allogeneictransplant” refers to transfer of cells or organs from a donor to arecipient, where the recipient is the same species as the donor.

“Autologous” refers to deriving from or originating in the same subjector patient. An “autologous transplant” refers to the harvesting andreinfusion or transplant of a subject's own cells or organs. Exclusiveor supplemental use of autologous cells can eliminate or reduce manyadverse effects of administration of the cells back to the host,particular graft versus host reaction.

“Chemically-defined” as used herein refers to culture media of knownchemical composition, both quantitatively and qualitatively, with nodeliberately added uncharacterized supplements, even though such amedium may contain trace contaminants in its components. Achemically-defined medium necessarily lacks animal serum, feeder cellssuch as stromal cells, and cell-based extracellular matrices derivedfrom, e.g., fibroblasts and the like.

“Committed myeloid progenitor cell” or “myeloid progenitor cell” refersto a multipotent or unipotent progenitor cell capable of ultimatelydeveloping into any of the terminally differentiated cells of themyeloid lineage, but which do not typically differentiate into cells ofthe lymphoid lineage. Hence, “myeloid progenitor cell” refers to anyprogenitor cell in the myeloid lineage. Committed progenitor cells ofthe myeloid lineage include oligopotent CMP, GMP, and MEP as definedherein, but also encompass unipotent erythroid progenitor, megakaryocyteprogenitor, granulocyte progenitor, and macrophage progenitor cells.Different cell populations of myeloid progenitor cells aredistinguishable from other cells by their differentiation potential, andthe presence of a characteristic set of cell markers.

“Committed lymphoid progenitor cell” or “lymphoid progenitor cell”refers to an oligopotent or unipotent progenitor cell capable ofultimately developing into any of the terminally differentiated cells ofthe lymphoid lineage, such as T cell, B cell, NK cell, or lymphoiddendritic cells, but which do not typically differentiate into cells ofthe myeloid lineage. As with cells of the myeloid lineage, differentcell populations of lymphoid progenitors are distinguishable from othercells by their differentiation potential, and the presence of acharacteristic set of cell markers.

“Common lymphoid progenitor cell” or “CLP” refers to an oligopotent cellcharacterized by its capacity to give rise to B-cell progenitors (BCP),T-cell progenitors (TCP), NK cells, and dendritic cells. Theseprogenitor cells have little or no self-renewing capacity, but arecapable of giving rise to T lymphocytes, B lymphocytes, NK cells, andlymphoid dendritic cells.

“Common myeloid progenitor cell” or “CMP” refers to a cell characterizedby its capacity to give rise to granulocyte/monocyte (GMP) progenitorcells and megakaryocyte/erythroid (MEP) progenitor cells. Theseprogenitor cells have limited or no self-renewing capacity, but arecapable of giving rise to myeloid dendritic, myeloid erythroid,erythroid, megakaryocytes, granulocyte/macrophage, granulocyte, andmacrophage cells.

“Congenic” refers to deriving from, originating in, or being members ofthe same species, where the members are genetically identical except fora small genetic region, typically a single genetic locus (i.e., a singlegene). A “congenic transplant” refers to transfer of cells or organsfrom a donor to a recipient, where the recipient is geneticallyidentical to the donor except for a single genetic locus.

“Cytokine” refers to compounds or compositions that in the natural stateare made by cells and affect physiological states of the cells thatproduce the cytokine autocrine factors) or other cells. Cytokine alsoencompasses any compounds or compositions made by recombinant orsynthetic processes, where the products of those processes haveidentical or similar structure and biological activity as the naturallyoccurring forms. Lymphokines refer to natural, synthetic, or recombinantforms of cytokines naturally produced by lymphocytes, including, but notlimited to, IL-1, IL-3, IL-4, IL-6, IL-11, and the like.

“Expansion” in the context of cells refers to increase in the number ofa characteristic cell type, or cell types, from an initial population ofcells, which may or may not be identical. The initial cells used forexpansion need not be the same as the cells generated from expansion.For instance, the expanded cells may be produced by growth anddifferentiation of the initial population of cells. Excluded from theterm expansion are limiting dilution assays used to characterize thedifferentiation potential of cells.

“Functional” in the context of cells refers to cells capable ofperforming or cells that retain the regular functions or activitiesassociated with the specified cell type, as identified by a definedfunctional assay or assays. For instance, a “functional GMP cell” is aprogenitor cell capable of ultimately differentiating into granulocytesand macrophages, where the terminally differentiated cells function asnormal granulocytes and macrophages.

“Graft-versus-host response” or “GVH” or “GVHD” refers to a cellularresponse that occurs when lymphocytes of a different MHC class areintroduced into a host, resulting in the reaction of the donorlymphocytes against the host.

“Granulocyte/macrophage progenitor cell” or “GMP” refers to a cellderived from common myeloid progenitor cells, and characterized by itscapacity to give rise to granulocyte and macrophage cells, but whichdoes not typically give rise to erythroid cells or megakaryocytes of themyeloid lineage.

“Growth factor” refers to a compound or composition that in the naturalstate affects cell proliferation, cell survival, and/or differentiation.A growth factor, while having the indicated effect on the cell, may alsoaffect other physiological process, such as secretion, adhesion,response to external stimuli, and the like. Although many growth factorsare made by cells, growth factors as used herein also encompass anycompound or composition made by recombinant or synthetic processes,where the product of those processes have identical or similar structureand biological activity as the naturally occurring growth factor.Examples of growth factors include epidermal growth factor (EGF),fibroblast growth factor (FGF), erythropoietin (EPO), thromobopoietin(TPO), stem cell factor (SCF), and flt-3 ligand (FL), and analogsthereof.

“Isolated” refers to a product, compound, or composition which isseparated from at least one other product, compound, or composition withwhich it is associated in its naturally occurring state, whether innature or as made synthetically.

“Hematopoietic stem cell” or “HSC” refers to a clonogenic, self-renewingpluripotent cell capable of ultimately differentiating into all celltypes of the hematopoietic system, including B cells T cells, NK cells,lymphoid dendritic cells, myeloid dendritic cells, granulocytes,macrophages, megakaryocytes, and erythroid cells. As with other cells ofthe hematopoietic system, HSCs are typically defined by the presence ofa characteristic set of cell markers. “Enriched” when used in thecontext of HSC refers to a cell population selected based on thepresence of a single cell marker, generally CD34+, while “purified” inthe context of HSC refers to a cell population resulting from aselection on the basis of two or more markers, preferably CD34+CD90+.

“Marker phenotyping” refers to identification of markers or antigens oncells for determining their phenotype (e.g., differentiation stateand/or cell type). This may be done by immunophenotyping, which usesantibodies that recognize antigens present on a cell. The antibodies maybe monoclonal or polyclonal, but are generally chosen to have minimalcrossreactivity with other cell markers. It is to be understood thatcertain cell differentiation or cell surface markers are unique to theanimal species from which the cells are derived, while other cellmarkers will be common between species. These markers definingequivalent cell types between species are given the same markeridentification even though there are species differences in structure(e.g., amino acid sequence). Cell markers include cell surfacesmolecules, also referred to in certain situations as celldifferentiation (CD) markers, and gene expression markers. The geneexpression markers are those sets of expressed genes indicative of thecell type or differentiation state. In part, the gene expression profilewill reflect the cell surface markers, although they may includenon-cell surface molecules.

“Megakaryocyte/erythroid progenitor cell” or “MEP” refers to a cellderived from common myeloid progenitor cells, and characterized by itscapacity to give rise to erythroid cells and megakaryocytes, but whichdoes not typically give rise to granulocytes, macrophages, or myeloiddendritic cells.

“Mismatched allogeneic” refers to deriving from, originating in, orbeing members of the same species having non-identical majorhistocompatability complex (MHC) antigens (i.e., proteins) as typicallydetermined by standard assays used in the art, such as serological ormolecular analysis of a defined number of MHC antigens. A “partialmismatch” refers to partial match of the MHC antigens tested betweenmembers, typically between a donor and recipient. For instance, a “halfmismatch” refers to 50% of the MHC antigens tested as showing differentMHC antigen type between two members. A “full” or “complete” mismatchrefers to all MHC antigens tested as being different between twomembers.

“Myeloablative” or “myeloablation” refers to impairment or destructionof the hematopoietic system, typically by exposure to a cytotoxic agentor radiation. Myeloablation encompasses complete myeloablation broughton by high doses of cytotoxic agent or total body irradiation thatdestroys the hematopoietic system. It also includes a less than completemyeloablated state caused by non-myeloablative conditioning. Thus,non-myeloablative conditioning is treatment that does not completelydestroy the subject's hematopoietic system.

“Neutropenia” refers to a lower than normal number of neutrophils andother polymorphonuclear leukocytes in the peripheral blood. Typically, aneutropenic condition is diagnosed based on the absolute neutrophilcount (ANC), which is determined by multiplying the percentage of bandsand neutrophils on a differential by the total white blood cell count.Clinically, an abnormal ANC is fewer than about 1500 cells per ml ofperipheral blood. The severity of neutropenia is categorized as mild foran ANC of 1000-1500 cells per ml, moderate for an ANC of 500-1000 cellsper ml, and severe for an ANC of fewer than 500 cells per ml.

“Self renewal” refers to the ability of a cell to divide and generate atleast one daughter cell with the identical (e.g., self-renewing)characteristics of the parent cell. The second daughter cell may committo a particular differentiation pathway. For example, a self-renewinghematopoietic stem cell divides and forms one daughter stem cell andanother daughter cell committed to differentiation in the myeloid orlymphoid pathway. A committed progenitor cell has typically lost theself-renewal capacity, and upon cell division produces two daughtercells that display a more differentiated (i.e., restricted) phenotype.

“Short term repopulating hematopoietic stem cells” or “ST-HSC” refers tohematopoietic stem cells that have limited, short term self-renewingcapacity, and are characterized by their capacity to differentiate intocells of the myeloid and lymphoid lineage. ST-HSC are distinguished fromlong-term repopulating (LT) HSCs by their limited length of self-renewalactivity in culture assays (e.g., approximately 8 weeks; see,Christensen, J. L. and Weissman, I. L., Proc. Natl. Acad. Sci. USA(2001)).

“Sorting” as it pertains to cells refers to separation of cells based onphysical characteristics (such as, e.g., elutriation or other size-basedtechniques) or presence of markers (such as sorting using side scatter(SSC) and forward scatter (FSC), or fluorescence activation cell sorting(FACS) using labeled antibodies), or analysis of cells based on presenceof cell markers, e.g., FACS without sorting, and including as wellimmunoabsorption techniques such as, e.g., magnetic cell separationsystems.

“Substantially pure cell population” refers to a population of cellshaving a specified cell marker characteristic and differentiationpotential that is at least about 50%, preferably at least about 75-80%,more preferably at least about 85-90%, and most preferably at leastabout 95% of the cells making up the total cell population. Thus, a“substantially pure cell population” refers to a population of cellsthat contain fewer than about 50%, preferably fewer than about 20-25%,more preferably fewer than about 10-15%, and most preferably fewer thanabout 5% of cells that do not display a specified marker characteristicand differentiation potential under designated assay conditions.

“Subject” or “patient” are used interchangeably and refer to, exceptwhere indicated, mammals such as humans and non-human primates, as wellas rabbits, rats, mice, goats, pigs, and other mammalian species.

“Syngeneic” refers to deriving from, originating in, or being members ofthe same species that are genetically identical, particularly withrespect to antigens or immunological reactions. These include identicaltwins having matching MHC types. Thus, a “syngeneic transplant” refersto transfer of cells or organs from a donor to a recipient who isgenetically identical to the donor.

“Thrombocytopenia” refers to a lower than normal platelet count,generally less than about 100×10⁹/L, which gives rise to increasedclotting time and increased risk of spontaneous bleeding, particularlyat platelet levels of about 10-50×10⁹/L or lower. The condition occurswhen platelets are lost from circulation at a faster rate than theirreplenishment by megakaryocytes. Thrombocytopenia may result from eitherfailure of platelet synthesis and/or increased rate of plateletdestruction.

“Xenogeneic” refers to deriving from, originating in, or being membersof different species, e.g., human and rodent, human and swine, human andchimpanzee, etc. A “xenogeneic transplant” refers to transfer of cellsor organs from a donor to a recipient where the recipient is a speciesdifferent from that of the donor.

6.2 Cell Types and Sources of Cells for Expansion

The cell types relevant to the present disclosure are those of thehematopoietic system, particularly hematopoieitc stem cells and cells ofthe myeloid lineage. Descriptions of cells herein will use those knownto the skilled artisan, with the understanding that these descriptionsreflect the current state of knowledge in the art and the invention isnot limited thereby to only those phenotypic markers described herein.

The hematopoietic stem cells (HSC) are pluripotent stem cells capable ofself-renewal and are characterized by their ability to give rise underpermissive conditions to all cell types of the hematopoietic system. HSCself-renewal refers to the ability of an HSC cell to divide and produceat least one daughter cell with the same self renewal anddifferentiation potential of a HSC; that is, cell division gives rise toadditional HSCs. Self-renewal provides a continual source ofundifferentiated stem cells for replenishment of the hematopoieticsystem. The marker phenotypes useful for identifying HSCs will be thosecommonly known in the art. For human HSCs, the cell marker phenotypespreferably include CD34⁺ CD38⁻ CD90(Thy1)⁺ Lin⁻. For mouse HSCs, anexemplary cell marker phenotype is Sca-1⁺ CD90⁺ (see, e.g., Spangrude,G. J. et al., Science 1:661-673 (1988)) or c-kit⁺ Thy^(lo) Lin⁻ Sca-1⁺(see, Uchida, N. et al., J. Clin. Invest. 101(5):961-966 (1998)).Alternative HSC markers such as aldehyde dehydrogenase (see Storms etal., Proc. Nat'l Acad. Sci. 96:9118-23 (1999) and AC133 (see Yin et al.,Blood 90:5002-12 (1997) may also find advantageous use.

HSCs give rise to committed lymphoid or myeloid progenitor cells. Asused herein committed myeloid progenitor cells refer to cell populationscapable of differentiating into any of the terminally differentiatedcells of the myeloid lineage. Encompassed within the myeloid progenitorcells are the common myeloid progenitor cells (CMP), a cell populationcharacterized by limited or non-self-renewal capacity but which iscapable of cell division to form granulocyte/macrophage progenitor cells(GMP) and megakaryocyte/erythroid progenitor cells (MEP). Non-selfrenewing cells refers to cells that undergo cell division to producedaughter cells, neither of which have the differentiation potential ofthe parent cell type, but instead generates differentiated daughtercells. The marker phenotypes useful for identifying CMPs include thosecommonly known in the art. For CMP cells of murine origin, the cellpopulation is characterized by the marker phenotype c-Kit^(high) (CD117)CD16^(low) CD34^(low) Sca-1^(neg) Lin^(neg) and further characterized bythe marker phenotypes FcγR^(lo) IL-7Rα^(neg) (CD127). The murine CMPcell population is also characterized by the absence of expression ofmarkers that include B220, CD4, CD8, CD3, Ter119, Gr-1 and Mac-1. ForCMP cells of human origin, the cell population is characterized byCD34⁺CD38⁺ and further characterized by the marker phenotypes CD123⁺(IL-3Rα) CD45RA^(neg). The human CMP cell population is alsocharacterized by the absence of cell markers CD3, CD4, CD7, CD8, CD10,CD11b, CD14, CD19, CD20, CD56, and CD234a. Descriptions of markerphenotypes for various myeloid progenitor cells are described in, forexample, U.S. Pat. Nos. 6,465,247 and 6,761,883; Akashi, Nature 404:193-197 (2000); all publications incorporated herein by reference intheir entirety.

Another committed progenitor cell of the myeloid lineage is thegranulocyte/macrophage progenitor cell (GMP). The cells of thisprogenitor cell population are characterized by their capacity to giverise to granulocytes (e.g., basophils, eisinophils, and neutrophils) andmacrophages. Similar to other committed progenitor cells, GMPs lackself-renewal capacity. Murine GMPs are characterized by the markerphenotype c-Kit^(hi) (CD117) Sca-1^(neg)FcγR^(hi) (CD16) IL-7Rγ^(neg)CD34^(pos). Murine GMPs also lack expression of markers B220, CD4, CD8,CD3, Gr-1, Mac-1, and CD90. Human GMPs are characterized by the markerphenotype CD34⁺ CD38⁺ CD123⁺ CD45RA⁺. Human GMP cell populations arealso characterized by the absence of markers CD3, CD4, CD7, CD8, CD10,CD11b, CD14, CD19, CD20, CD56, and CD235a.

Where relevant to the discussion, the megakaryocyte/erythroid progenitorcells (MEP), which are derived from the CMPs, are characterized by theircapability of differentiating into committed megakaryocyte progenitorand erythroid progenitor cells. Mature megakaryocytes are polyploidcells that are precursors for formation of platelets, a developmentalprocess regulated by thrombopoietin. Erythroid cells are formed from thecommitted erythroid progenitor cells through a process regulated byerythropoietin, and ultimately differentiate into mature red bloodcells. Murine MEPs are characterized by cell marker phenotype c-Kit^(hi)and IL-7Rα^(neg) and further characterized by marker phenotypesFcγR^(lo) and CD34^(low). Murine MEP cell populations are alsocharacterized by the absence of markers B220, CD4, CD8, CD3, Gr-1, andCD90. Another exemplary marker phenotype for mouse MEPs is c-kit^(high)Sca-1^(neg) Lin^(neg/low) CD16^(low) CD34^(low). Human MEPs arecharacterized by marker phenotypes CD34⁺ CD38⁺ CD123^(neg) CD45RA^(neg).Human MEP cell populations are also characterized by the absence ofmarkers CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, andCD235a.

Further restricted progenitor cells in the myeloid lineage are thegranulocyte progenitor, macrophage progenitor, megakaryocyte progenitor,and erythroid progenitor. Granulocyte progenitor cells are characterizedby their capability to differentiate into terminally differentiatedgranulocytes, including eosinophils, basophils, neutrophils. The GPstypically do not differentiate into other cells of the myeloid lineage.With regards to the megakaryocyte progenitor cell (MKP), these cells arecharacterized by their capability to differentiate into terminallydifferentiated megarkaryocytes but generally not other cells of themyeloid lineage (see, e.g., WO 2004/024875).

For the lymphoid lineage, a “committed lymphoid progenitor cell” refersto a cell capable of differentiating into any of the terminallydifferentiated cells of the lymphoid lineage. Encompassed within thelymphoid progenitor cells are the common lymphoid progenitor cells(CLP), a cell population characterized by limited or non-self-renewalcapacity but which is capable of cell division to form T lymphocyte andB lymphocyte progenitor cells, NK cells, and lymphoid dendritic cells.The marker phenotypes useful for identifying CLPs will be those commonlyknown in the art. For CLP cells of mouse, the cell population ischaracterized by the presence of markers as described in Kondo, M. etal., Cell 91:661-672 (1997), while for human CLPs, a marker phenotype ofCD34⁺ CD38⁺ CD10⁺ IL7R+ may be used (Galy, A et al., Immunity, 3:459-473(1995); Akashi, K. et al., Int. J. Hematol. 69(4):217-226 (1999));publications incorporated herein by reference.

A summary of preferred murine cell surface markers is provided in Table1 below, where an approximate indication of staining levels is shown bythe cell colors in the tables: an asterisk indicates no staining, acaret indicates low level staining and a dagger indicates intermediateor high staining.

TABLE 1 HSC CD117† CD90.1{circumflex over ( )} Lin1* Sca-1† * * CLPCD117† CD90.1* Lin1* Sca-1† CD127† * MP CD117† Sca-1* Lin2* * * * CMPCD117† Sca-1* Lin2* CD16/32{circumflex over ( )} CD34† CD9* GMP CD117†Sca-1* Lin2* CD16/32† CD34† CD9* MEP CD117† Sca-1* Lin2*CD16/32{circumflex over ( )} CD34{circumflex over ( )} CD9* MKP CD117†Sca-1* Lin2* CD16/32{circumflex over ( )} CD34{circumflex over ( )} CD9†Lin1: CD3, CD4, CD5, CD8, B220, Gr-1, CD11b, TER119 Lin2: CD3, CD4, CD5,CD8, B220, Gr-1, CD90.1, CD127, TER119

A summary of preferred human cell surface markers is provided in Table 2below, where an approximate indication of staining levels is shown bythe cell colors in the tables: an asterisk indicates no staining, acaret indicates low level staining and a dagger indicates intermediateor high staining.

TABLE 2 HSC CD34† CD90† Lin1* * * CLP CD34† CD127† CD10† CD38/CD90* Lin*MP CD34† CD90* Lin2* * * CMP CD34† CD90* Lin2* CD45RA† CD123{circumflexover ( )} GMP CD34† CD90* Lin2* CD45RA† CD123† MEP CD34† CD90* Lin2*CD45RA{circumflex over ( )} CD123{circumflex over ( )} Lin1: CD2, CD3,CD7, CD8, CD10, CD11b, CD14, CD19, CD56, CD235a Lin2a: CD2, CD3, CD4,CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a Lin2b: CD10,CD11b, CD14, CD19, CD235a

Numerous other suitable cell surface markers are presently known to theskilled artisan, or will be identified and characterized in due course,and such markers will find advantageous use in the methods andcompositions described herein. For instance, several additionalpotential murine markers have recently been identified for the variousmyeloid progenitor cell populations based on array analysis of mRNAexpression. See, e.g., Iwasaki-Arai, et al. J. Exp. Med. 197:1311-1322(2003); Akashi, et al. Nature 404:193-197 (2000); Miyamoto, et al. Dev.Cell 3:137-147 (2002); Traver, et al. Blood 98:627-635 (2001); Akashi,et al. Blood 101:383-390 (2003); Terskikh, A., et al. Blood102:102:94-101 (2003). Based on this same type of mRNA expressionanalysis, additional cell surface markers such as CD110, CD114, CD116,CD117, CD127, and CD135 may also find use for isolating one or more ofthe identified myeloid progenitor subpopulations in humans, as describedin Manz, et al. Proc Natl Acad Sci USA 99:11872-11877 (2002).

For the methods described herein, the cells for expansion will be cellscapable of ultimately differentiating into cells of the myeloid lineage,i.e., granulocytes, macrophages, megakaryocytes, erythroid cells, and/ormyeloid dendritic cells. These include, among others, HSCs, andcommitted myeloid progenitor cells CMPs, GMPs, and MEPs. These cellswill have the relevant characteristics, particularly differentiationpotential and cell marker characteristics described above. In oneembodiment, the initial cells for the expansion comprise cells withmarker phenotype CD34⁺. Because the CD34 marker is found in differentprogenitor cell types, the initial cell population for expansion can bea mixture of progenitor cells expressing CD34. In another embodiment,the cells are cells comprising the cell marker phenotype Sca-1^(pos), acell marker found in mouse, and other rodents. Selection for Sca-1^(pos)cells will also result in a mixture of cells displaying the cell markerphenotype, although it will primarily select for HSCs because the mousecommitted myeloid progenitor cells are Sca-1^(neg). Thus, in otherembodiments for expansion of rodent myeloid cells, the cell markerphenotype of Lin^(neg/low) is used, which includes HSCs, CMPs, and GMPs.

In a further aspect, the initial cells for expansion are isolated cells.These include isolated HSCs, which under the presence of the indicatedmixture of cytokines and growth factors, develop into CMPs that furtherexpand into other progenitor cells of the myeloid lineage. In anotherembodiment, the initial cells for expansion are CMPs with thecharacteristic differentiation potential and cell marker phenotypes asdescribed above. CMPs may have limited self-renewal capacity, and thuscan expand to generate additional CMPs for a limited number of cellsdivisions while also differentiating into GMPs and MEPs.

Cells for expansion can be obtained from a variety of sources, includingbone marrow, peripheral blood, cord blood, and other sources known toharbor hematopoietic and myeloid progenitor cells, including liver,particularly fetal liver. Peripheral and cord blood is a rich source ofHSCs and progenitor cells. Cells are obtained using methods known andcommonly practiced in the art. For example, methods for preparing bonemarrow cells are described in Sutherland et al., Bone Marrow Processingand Purging: A Practical Guide (Gee, A. P. ed.), CRC Press Inc. (1991)).Umbilical cord blood or placental cord blood is typically obtained bypuncture of the umbilical vein, in both term or preterm, before or afterplacental detachment (see, e.g., Turner, C. W. et al., Bone MarrowTransplant. 10:89 (1992); Bertolini, F. et al., J. Hematother. 4:29(1995)). HSCs and myeloid progenitor cells may also be obtained fromperipheral blood by leukapheresis, a procedure in which blood drawn froma suitable subject is processed by continuous flow centrifugation (e.g.,Cobe BCT Spectra blood cell separators) to remove white blood cellswhile the other blood components are returned to the donor. Another typeof isolation procedure is centrifugation through a medium of varyingdensity, such as Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway,N.J.).

The cells are derived from any animal species with a hematopoieticsystem, as generally described herein. Preferably, suitable animals willbe mammals, including, by way of example and not limitation, rodents,rabbits, canines, felines, pigs, horses, cows, primates (e.g., human),and the like. The cells for the expansion may be obtained from a singlesubject, or a plurality of subjects. A plurality refers to at least two(e.g., more than one) donors. When cells obtained are from a pluralityof donors, their relationships may be syngeneic, allogenenic, orxenogeneic, as defined herein. A preferred embodiment of the presentdisclosure is directed to a mixture of allogeneic myeloid progenitorcells obtained by the expansion methods herein, as further describedbelow. The allogeneic cells may be expanded separately and the cellsmixed following expansion, or the cells mixed prior to expansion, asfurther discussed below.

Where applicable, stem cells and progenitor cells may be mobilized fromthe bone marrow into the peripheral blood by prior administration ofcytokines or drugs to the subject (see, e.g., Lapidot, T. et al., Exp.Hematol. 30:973-981 (2002)). Cytokines and chemokines capable ofinducing mobilization include, by way of example and not limitation,granulocyte colony stimulating factor (G-CSF), granulocyte macrophagecolony stimulating factor (GM-CSF), erythropoietin (Kiessinger, A. etal., Exp. Hematol. 23:609-612 (1995)), stem cell factor (SCF), AMD3100(AnorMed, Vancouver, Canada), interleukin-8 (IL-8), and variants ofthese factors (e.g., pegfilgastrim, darbopoietin). Combinations ofcytokines and/or chemokines, such as G-CSF and SCF or GM-CSF and G-CSF,can act synergistically to promote mobilization and may be used toincrease the number of HSC and progenitor cells in the peripheral blood,particularly for subjects who do not show efficient mobilization with asingle cytokine or chemokine (Morris, C. et al., J. Haematol.120:413-423 (2003)).

Cytoablative agents can be used at inducing doses (i.e., cytoreductivedoses) to also mobilize HSCs and progenitor cells, and are useful eitheralone or in combination with cytokines. This mode of mobilization isapplicable when the subject is to undergo myeloablative treatment, andis carried out prior to the higher dose chemotherapy. Cytoreductivedrugs for mobilization, include, among others, cyclophosphamide,ifosfamide, etoposide, cytosine arabinoside, and carboplatin (Montillo,M. et al., Leukemia 18:57-62 (2004); Dasgupta, A. et al., J. InfusionalChemother. 6:12 (1996); Wright, D. E. et al., Blood 97:(8):2278-2285(2001)).

The cells for expansion may also be subjected to further selection andpurification, which can include both positive and negative selectionmethods, to obtain a substantially pure population of cells. In oneaspect, fluorescence activated cell sorting (FACS), also referred to asflow cytometry, is used to sort and analyze the different cellpopulations. Cells having the cellular markers specific for HSC or aprogenitor cell population are tagged with an antibody, or typically amixture of antibodies, that bind the cellular markers. Each antibodydirected to a different marker is conjugated to a detectable molecule,particularly a fluorescent dye that can be distinguished from otherfluorescent dyes coupled to other antibodies. A stream of tagged or“stained” cells is passed through a light source that excites thefluorochrome and the emission spectrum from the cells detected todetermine the presence of a particular labeled antibody. By concurrentdetection of different fluorochromes, also referred to in the art asmulticolor fluorescence cell sorting, cells displaying different sets ofcell markers may be identified and isolated from other cells in thepopulation. Other FACS parameters, including, by way of example and notlimitation, side scatter (SSC), forward scatter (FSC), and vital dyestaining (e.g., with propidium iodide) allow selection of cells based onsize and viability. FACS sorting and analysis of HSC and progenitorcells is described in, among others, U.S. Pat. Nos. 5,137,809,5,750,397, 5,840,580; 6,465,249; Manz, M. G. et al., Proc. Natl. Acad.Sci. USA 99:11872-11877 (2002); and Akashi, K. et al., Nature404(6774):193-197 (2000)). General guidance on fluorescence activatedcell sorting is described in, for example, Shapiro, H. M., PracticalFlow Cytometry, 4th Ed., Wiley-Liss (2003) and Ormerod, M. G., FlowCytometry: A Practical Approach, 3rd Ed., Oxford University Press(2000).

Another method of isolating the initial cell populations uses a solid orinsoluble substrate to which is bound antibodies or ligands thatinteract with specific cell surface markers. In immunoadsorptiontechniques, cells are contacted with the substrate (e.g., column ofbeads, flasks, magnetic particles) containing the antibodies and anyunbound cells removed. Immunoadsorption techniques can be scaled up todeal directly with the large numbers of cells in a clinical harvest.Suitable substrates include, by way of example and not limitation,plastic, cellulose, dextran, polyacrylamide, agarose, and others knownin the art (e.g., Pharmacia Sepharose 6 MB macrobeads). When a solidsubstrate comprising magnetic or paramagnetic beads is used, cells boundto the beads can be readily isolated by a magnetic separator (see, e.g.,Kato, K. and Radbruch, A., Cytometry 14(4):384-92 (1993); CD34⁺ directisolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany). Affinitychromatographic cell separations typically involve passing a suspensionof cells over a support bearing a selective ligand immobilized to itssurface. The ligand interacts with its specific target molecule on thecell and is captured on the matrix. The bound cell is released by theaddition of an elution agent to the running buffer of the column and thefree cell is washed through the column and harvested as a homogeneouspopulation. As apparent to the skilled artisan, adsorption techniquesare not limited to those employing specific antibodies, and may usenonspecific adsorption. For example, adsorption to silica is a simpleprocedure for removing phagocytes from cell preparations.

FACS and most batch wise immunoadsorption techniques can be adapted toboth positive and negative selection procedures (see, e.g., U.S. Pat.No. 5,877,299). In positive selection, the desired cells are labeledwith antibodies and removed away from the remaining unlabeled/unwantedcells. In negative selection, the unwanted cells are labeled andremoved. Another type of negative selection that can be employed is useof antibody/complement treatment or immunotoxins to remove unwantedcells.

It is to be understood that the purification of cells also includescombinations of the methods described above. A typical combination maycomprise an initial procedure that is effective in removing the bulk ofunwanted cells and cellular material, for example leukapharesis. Asecond step may include isolation of cells expressing a marker common toone or more of the progenitor cell populations by immunoadsorption onantibodies bound to a substrate. For example, magnetic beads containinganti-CD34 antibodies are able to bind and capture HSC, CMP, and GMPcells that commonly express the CD34 antigen. An additional stepproviding higher resolution of different cell types, such as FACSsorting with antibodies to a set of specific cellular markers, can beused to obtain substantially pure populations of the desired cells.Another combination may involve an initial separation using magneticbeads bound with anti-CD34 antibodies followed by an additional round ofpurification with FACS.

Determining the differentiation potential of cells, and thus the type ofstem cells or progenitor cells isolated, is typically conducted byexposing the cells to conditions that permit development into variousterminally differentiated cells. These conditions generally comprise amixture of cytokines and growth factors in a culture medium permissivefor development of the myeloid or lymphoid lineage. Colony formingculture assays rely on culturing the cells in vitro via limitingdilution and assessing the types of cells that arise from theircontinued development. A common assay of this type is based onmethylcellulose medium supplemented with cytokines (e.g., MethoCult,Stem Cell Technologies, Vancouver, Canada; Kennedy, M. et al., Nature386:488-493 (1997)). Cytokine and growth factor formulations permissivefor differentiation in the hematopoietic pathway are described in Manzet al., Proc. Natl Acad. Sci. USA 99(18):11872-11877 (2002); U.S. Pat.No. 6,465,249; and Akashi, K. et al., Nature 404(6774):193-197 (2000)).Cytokines include SCF, FLT-3 ligand, GM-CSF, IL-3, TPO, and EPO. Anotherin vitro assay is long-term culture initiating cell (LTC-IC) assay,which typically uses stromal cells to support hematopoiesis (see, e.g.,Ploemacher, R. E. et al., Blood. 74:2755-2763 (1989); and Sutherland, H.J. et al., Proc. Natl. Acad. Sci. USA 87:3745 (1995)).

Another type of assay suitable for determining the differentiationpotential of isolated cells relies upon in vivo administration of cellsinto a host animal and assessment of the repopulation of thehematopoietic system. The recipient is immunocompromised orimmunodeficient to limit rejection and permit acceptance of allogeneicor xenogeneic cell transplants. A useful animal system of this kind isthe NOD/SCID (Pflumio, F. et al., Blood 88:3731 (1996); Szilvassym S. J.et al., “Hematopoietic Stem Cell Protocol,” in Methods in MolecularMedicine, Humana Press (2002); Greiner, D. L. et al., Stem Cells16(3):166-177 (1998); Piacibello, W. et al., Blood 93:(11):3736-3749(1999)) or Rag2 deficient mouse (Shinkai, Y. et al., Cell 68:855-867(1992)). Cells originating from the infused cells are assessed byrecovering cells from the bone marrow, spleen, or blood of the hostanimal and determining presence of cells displaying specific cellularmarkers, (i.e., marker phenotyping) typically by FACS analysis.Detection of markers specific to the transplanted cells permitsdistinguishing between endogenous and transplanted cells. For example,antibodies specific to human forms of the cell markers (e.g., HLAantigens) identify human cells when they are transplanted into suitableimmunodeficient mouse (see, e.g., Piacibello. W. et al., supra).

The initial populations of cells obtained by the methods above are useddirectly for expansion or frozen for use at a later date. A variety ofmediums and protocols for freezing cells are known in the art.Generally, the freezing medium will comprise DMSO from about 5-10%,10-90% serum albumin, and 50-90% culture medium. Other additives usefulfor preserving cells include, by way of example and not limitation,disaccharides such as trehalose (Scheinkonig, C. et al., Bone MarrowTransplant. 34(6):531-6 (2004)), or a plasma volume expander, such ashetastarch hydroxyethyl starch). In some embodiments, isotonic buffersolutions, such as phosphate-buffered saline, may be used. An exemplarycryopreservative composition has cell-culture medium with 4% HSA, 7.5%dimethyl sulfoxide (DMSO), and 2% hetastarch. Other compositions andmethods for cryopreservation are well known and described in the art(see, e.g., Broxmeyer, H. E. et al., Proc. Natl. Acad. Sci. USA100(2):645-650 (2003)). Cells are preserved at a final temperature ofless than about −135° C.

6.3 Ex Vivo Expansion of Myeloid Progenitor Cells

The initial population of cells obtained above is expanded ex vivo inculture by contacting the cells with a medium having a cytokine andgrowth factor mixture permissive for expansion of myeloid progenitorcells. Cytokines in their natural context are typically proteins made bycells that modulate a cell's physiological state, whether the cell isanother cell or the cell producing the cytokine. Cytokines made bylymphocytes are often described as lymphokines (IL), but are cytokinesas defined herein. Cytokines typically act via cellular receptors on thecells modulated by the cytokine. Likewise, growth factors in theirnatural context are also compounds typically made by cells, affectingthe proliferation and differentiation of cells, whether the cell isanother cell or the cell producing the growth factor. Like cytokines,growth factors generally act on cells via receptors. Reference to acytokine or growth factor in the present disclosure, however, is notmeant to be exclusionary since certain cytokines have effects onproliferation and differentiation of cells similar to growth factors.Hence, the descriptions of specific cytokines and growth factors hereinreflect the state of knowledge in the art and are not meant to limit thescope of this disclosure.

For the expansion methods herein, cytokines and growth factors arechosen to expand populations of committed myeloid progenitor cells, suchas CMP, GMP, and MEP cells. Since these cells have limited or noself-renewing capacity, the culture conditions are chosen to supportdivision of cells that develop into these myeloid cells while limitingor minimizing growth and expansion of other cell types that are notcommitted myeloid progenitors.

Accordingly, cytokines for expansion conditions are generally selectedfrom IL-1 (i.e., IL-1β), IL-3, IL-6, IL-11, G-CSF, CM-CSF, and analogsthereof. Forms of the cytokines are naturally occurring products,recombinant products, variants, or modified forms having similarbiological activity as the naturally occurring forms such as, e.g.,peptide mimetics. The cytokines may also be selected from the group offusion proteins or engineered cytokines, suitable non-limiting examplesinclude PIXY321 (Curtis, B. M., et al. Proc. Natl. Acad. Sci. U.S.A.1991 88.5809-5813) a synthetic hybrid protein of GM-CSF and IL-3,Epo-IL-3 (Lu, L., et al. Exp. Hematol. 1995 23. 1130-1134), IL-2-IL-6(Zhao. C., et al. Stem Cells 1994. 12. 1130-1134).

Source of the cytokines are chosen to be active on the cells used forexpansion, and thus will generally reflect the origin of the initialcells used for expansion. For example, if the progenitor cells are ofhuman origin, human forms of the cytokine, either natural orrecombinant, are used. Accordingly, in one embodiment, the cytokines arerecombinant human rhuIL-1, rhuIL-1β), rhuIL-3, rhuIL-6, rhuIL-11,rhuG-CSF, rhuGM-CSF, and analogs thereof. However, the associationbetween the form of the cytokine and the origin of cells need not berigorous. For instance, human IL-6 is able to elicit effects in mouseand rat cells, although mouse IL-6 has no effect on human cells. Thistype of crossactivity will be apparent to the skilled artisan and can bereadily tested by known methods. The structure and function of thespecified cytokines will refer to the descriptions below, which reflectthe state of knowledge in the art.

IL-1 is a group of cytokines that has important roles in the up- anddown-regulation of acute inflammation (e.g., activation of endothelialcells and lymphocytes), bone formation and remodeling, insulinsecretion, and fever induction. IL-1 family of cytokines shares anoverall structural similarity, being composed of β-barrel with a pseudothree fold axis (see, e.g., Priestle, J. P. et al., Proc Natl Acad SciUSA 86, 9667-71 (1989)). Pertinent to the methods herein is IL-1β, whichis typically secreted by macrophages along with IL-1a. The two agonistsare derived by enzymatic cleavage of precursor proteins (pro-IL-1a andpro-IL-1β), and exert their physiological effects by binding to IL-1receptors. Amino acid sequences and their corresponding nucleic acidsequences for IL-13 are known from various sources, including, by way ofexample and not limitation, murine (Telford, J. L. et al., NucleicAcids, Res. 14(24):9955-9963 (1986)); rabbit (Young P R and SylvesterD., Protein Eng. 2(7):545-51 (1989)); rat (Accession No. NP 113700[gi:13928692]); porcine (Huether, M. J, et al., Gene 129(2):285-289(1993)); bovine (Leong S. R., Nucleic Acids Res. 16:9054-9054(1988));feline (Daniel, S. L. et al., Anim. Biotechnol. 3:117-121 (1992));equine (Howard, R. D. et al., Am. J. Vet. Res. 59:704-711(1998)); human(March, C. J. et al., Nature 315:641 (1985); and recombinant human(Meyers, C. et al., J. Biol. Chem. 262(23):11176-11181 (1987)). Variantsof IL-1β are described in Boraschi, D. et al., Frontiers in Bioscience1:270-308 (1995)). Various recombinant forms are also availablecommercially (see, e.g., human IL-1β, Promega, Madison, Wis., USA;murine IL-1β, Stem Cell Technologies, Vancouver, BC, Canada; and ratIL-1β, Chemicon Int., Temacula, Calif., USA). Variants of IL-1β aredescribed in, among others, Gronenborn, A. M. et al., Eur. J. Biochem.161(1):37-43 (1986); Antoni, Ge et al., J. Immunol. 137(10):3201-4(1986); Palaszynski, E. W., Biochem. Biophys. Res. Commun. 147(1):204-11(1987); and Huang, J. J. et al, FEBS Lett. 223(2):294-8 (1987)).

IL-3, also know as multi-CSF, is a multilineage cell cytokine/growthfactor secreted by lymphocytes, epithelial cells, and astrocytes, thatstimulates the clonal proliferation and differentiation of various typesof blood and tissue cells, particularly the differentiation and functionof granulocytes and macrophages. It is considered one of thehematopoietic colony stimulating factors (see, e.g., Wagemaker, G. etal., Biotherapy 2(4):337-45 (1990)). Amino acid and nucleic acidsequences for IL-3 have been identified from various organisms,including, among others, murine (Fung M.-C. et al., Nature307:233-237(1984)); rat (Cohen, D. R. et al. Nucleic Acids Res.14:3641-3658(1986); sheep (McInnes C. J. et al., Gene 139:289-290(1994)); bovine (Mwangi S. M. et al., Gene 162:309-312(1995));chimpanzee/monkeys (Burger H. et al., Biochim. Biophys. Acta1217:195-198(1994)); and human (Yang Y.-C. et al., Cell 47:3-10 (1986);Otsuka T. et al., J. Immunol. 140:2288-2295(1988)). Variants of IL-3 aredescribed in Lopez, A. F. et al., Proc Natl Acad Sci USA 89(24):11842-6(1992); Barry, S. C. et al., J Bio.l Chem. 269(11):8488-92 (1994); andOlins, P. O. et al., J Biol Chem. 270(40):23754-60 (1995)).

IL-6 is known as B-cell stimulatory factor 2 (BSF-2) and interferon-β2,and is involved in regulating differentiation of B cells intoimmunoglobulin secreting cells, induction of myeloma/plasmacytomagrowth, and nerve cell differentiation. IL-6 binding to IL-6 receptorsinduces formation of a multisubunit complex containing protein GP130,which is common to the class I cytokine receptor superfamily. IL-6appears to have a common structure composed of a four helical bundle,where the helical faces interact with the receptor. Amino acid andnucleic acid sequences for IL-6 have been identified for, among others,mouse (Chiu, C. P. et al., Proc. Natl. Acad. Sci. USA. 85(19):7099-103(1988); rat (Northemann, W. et al., J. Biol. Chem. 264(27):16072-82(1989); rabbit (Perkins, H. D. et al., Cytokine 12(6):555-65 (2000));sheep (Ebrahimi, B. et al., Cytokine. 7(3):232-236 (1995)); bovine(Droogmans, L. et al., DNA Seq. 2(6):411-3 (1992)); equine (Swiderski,C. E. et al., Vet Immunol Immunopathol. 77(3-4):213-20 (2000)); andhuman (Hirano T. et al., Nature 324: 73-76 (1986)). Variants of IL-6 aredescribed in Dagan, S. et al., Protein Expr. Purif. 3(4):290-4 (1992);Zhang, J. G. et al., Eur J Biochem. 207(3):903-13 (1992); and Skelly, S.M. et al., J Biotechnol. 34(1):79-86 (1994). Recombinant forms aredescribed in Stoyan, T. et al., Eur J Biochem. 216(1):239-45 (1993));Orita, T. et al., J Biochem (Tokyo) 115(2):345-50 (1994)), and are alsocommercially available.

IL-11 belongs to the IL-6 group of structurally and functionally relatedcytokines, which, as noted above, uses the transmembrane glycoproteingp130 to exert its physiological activity. IL-11 is also known asadipogenesis inhibitor factor (AGIF) and oprelvekin. IL-11 actssynergistically with other cytokines and growth factors to stimulateproliferation and differentiation of stem cells into committedprogenitor cells and to promote megakaryopoiesis and thrombopoiesis.Opposing effects of IL-11 are seen in vivo and in vitro in that it canenhance engraftment in vivo while in vitro, IL-11 can maintain primitivepopulation of stem cells. Being a class 1 cytokine, IL-11 is alsobelieved to comprise a four helical bundle structure. Amino acid andnucleic acid sequences of IL-11 have been identified for, among others,murine (Morris, J. C. et al., Exp. Hematol. 24:1369 (1996); primate(Paul, S. R. et al., Proc. Natl. Acad. Sci. USA. 87(19):7512-6 (1990);and human (Ohsumi, J. et al., FEBS Lett. 288:13 (1991)). Recombinantforms and variants of IL-11 are described in Miyadai, K. et al., Biosci.Biotechnol. Biochem. 60(3):541-2 (1996); Tacken, I. et al., Eur JBiochem. 265(2):645-55 (1999)).

G-CSF or granulocyte-colony stimulating factor acts to induce the bonemarrow to produce granulocytes, and promote the survival, proliferation,differentiation and function of neutrophil granulocyte progenitor cellsand mature neutrophils. It is produced by a number of different celltypes, such as endothelial cells and macrophages. Although naturallyoccurring as a glycoprotein, G-CSF in its non-glycosylated form madethrough recombinant techniques is fully active. Structurally, G-CSF isrelated to the class 1 cytokine family, as indicated by the presence ofa four-alpha-helix bundle (Hill, C. et al., Proc Natl Acad Sci USA90(11):5167-71 (1993); Lovejoy, B. et al., J Mol Biol. 234(3):640-53(1993). Amino acid and nucleic acid sequences for G-CSF have beenidentified for, among others, murine (Tsuchiya, M. et al., Proc NatlAcad Sci USA. 83(20):7633-7 (1986); rat (Han, S. W. et al., Gene175(1-2):101-4 (1996)); bovine (Heidari, M. and Kehrli, M. E., Vet.Immunol. Immunopathol. 73(2):183-91 (2000); sheep (O'Brien, P. M., DNASeq. 4(5):339-42 (1994)); feline (Dunham, S. P. and Onions, D. E.,Cytokine 14(6):347-51 (2001)); porcine (Kulmburg, P. et al., Gene197(1-2):361-5 (1997)); and human (Nagata, S. et al., EMBO J. 5:575-581(1986)). Recombinant forms and variants of G-CSF are described in Lu, H.S. et al., Arch Biochem Biophys. 268(1):81-92 (1989); Kuga, T. et al.,Biochem Biophys Res Commun. 159(1):103-11 (1989); and Fujii, I. et al.,FEBS Lett. 410(2-3):131-5 (1997)), and commercially available under thetrade name filgrastim; lenograstim; pluripoietin, Neupogen®, granulokine(Amgen, Thousand Oaks, Calif., USA), and granocyte (Rhone-Poulenc).

GM-CSF or granulocyte-macrophage colony stimulating factor, also knownas colony stimulating factor 2 stimulates growth and differentiation ofhematopoietic precursor cells from various lineages, includinggranulocytes, macrophages, eosinophils and erythrocytes. It is also partof the class 1 cytokine family, having a four helical bundle structure,and exerts its physiological effect by binding to granulocyte-macrophagecolony stimulating factor receptor. Amino acid and nucleic acidsequences known for GM-CSF include, among others, mouse (Gough, N. M. etal., Nature 309:763-767(1984); sheep (McInnes, C. J. and Haig, M. C. K.,Gene 105:275-279(1991); bovine (Maliszewski, C. R., Mol. Immunol.25:843-850(1988)); and human (Cantrell, M. A. et al., Proc. Natl. Acad.Sci. USA 82:6250-6254(1985); Lee, F. et al., Proc Natl Acad Sci USA.82(13):4360-4 (1985)). Recombinant forms and variants of GM-CSF aredescribed in DeLamarter, J. F. et al., EMBO J. 4(10):2575-81 (1985);Shanafelt A. B. and Kastelein, R. A., Proc Natl Acad Sci USA.86(13):4872-6 (1989), and are available commercially under the tradenamemolgramostin and sargramostim.

The growth factors for purposes of expansion are selected from stem cellfactor (SCF or SF), FLT-3 ligand (FL), thrombopoietin (TPO),erythropoietin (EPO), and analogs thereof. As with the cytokines, growthfactor forms are either naturally occurring products or are recombinantforms having similar biological activity as the naturally occurringfactors. Accordingly, in one embodiment, the growth factors arerecombinant human rhuSCF, rhuFL, rhuTPO, rhuEPO, and analogs thereof. Aswith the selection of cytokines, growth factors are chosen to be activeon the cells used for expansion, and thus will generally reflect theorigin of the initial cells, although the association need not be arigorous one, as noted above. For instance, rat and mouse SCF are activeon human cells, but the human protein is much less active on mouse orrat cells. This type of crossactivity will be apparent to the skilledartisan and can be readily tested by known methods. The structure andfunction of the specified growth factors will refer to the descriptionsthat follow, which reflect the state of knowledge in the art and are notmeant to be limiting.

SCF, also known as c-kit ligand, mast cell growth factor, or Steelfactor, acts on multiple levels of the hematopoietic hierarchy topromote cell survival, proliferation, differentiation, adhesion andfunctional activation in combination with other cytokines. It is ofparticular importance in the myeloid lineages, particularly on thedevelopment of mast cells, but also acts on multipotent stem andprogenitor cells, megakaryocytes, and a subset of lymphoid progenitor(Broudy, V. C., Blood 90(4):1345-1364 (1997)). SCF exerts its biologicaleffects by binding to its receptor, C-KIT. Naturally occurring SCF issynthesized by bone marrow stromal cells as either a transmembrane formor a soluble form, both of which are biologically active. The overallstructure of SCF has an anti-parallel four-helix bundle fold (Zhang, Z.et al., Proc. Natl. Acad. Sci. USA 97(14):7732-7 (2000)). Amino acid andnucleic acid sequences known for SCF include, among others, murine(Lyman, S. D. et al., Cell 75(6):1157-67 (1993)), rat (Martin, F. H. etal., Cell 63(1):203-11 (1990)); feline(Dunham, S. P. and Onions, D. E.,DNA Seq. 6(4):233-7 (1996); sheep (McInnes, C. J. et al., Cytokine11(4):249-56 (1999)); canine (Shin, I. S. et al., J Vet Sci. 2(3):159-66(2001)); and human (Martin, F. H. et al., supra). Recombinant SCF andvariants are described in Jones, M. D. et al., J. Biol. Chem. 271:11301(1996); Lu, H. S. et al., J. Biol. Chem. 271:11309 (1996); Langley, K.E. et al., Arch. Biochem. Biophys. 295:21 (1992); Lev, S. et al., MolCell Biol. 13(4):2224-34 (1993); and Langley, K. E. et al., Arch.Biochem. Biophys. 311:(1):55-61 (1994).

“FLT-3 ligand”, also known as “FL” or “SL cytokine” or “FMS relatedtyrosine kinase 3 ligand” is a factor that binds to flt-3 receptor(also, “ACD135” or “Aflk2”), a tyrosine kinase receptor moleculegenerally found on hematopoietic stem cells and primitive progenitorcells, including CD34+ cells. It synergizes with other factors such asCD117 (c-kit) to stimulate hematopoietic stem cell proliferation invitro and stimulate expansion and mobilization of progenitor cells invivo (Lyman, S. D. and Williams, D. E., Curr. Opin. Hematol. 2(3):177-81(1995)). Both the full-length FLT-3 ligand (composed of an extracellulardomain, a transmembrane domain and an intracellular domain) and asoluble extracellular domain are biologically active (Lyman, S. D. etal., Cell 75:1157 (1993); Lyman, S. D. et al., Blood 83:2795 (1994)).Preferably, the FLT-3 ligand is the soluble form that contains the aminoacid sequence of the full-length extracellular domain. Structure ofsoluble FLT-3 ligand reveals the presence of two short chainalpha-helical bundles, similar to SCF and G-CSF (Sawides, S. N. et al.,Nat. Struct. Biol. 7(6):486-91 (2000)). Amino acid and nucleic acidsequences of FL have been identified for, among others, murine (Rosnet,O. et al., Oncogene. 6(9):1641-50 (1991); feline (Yang S. and Sim, G.K., DNA Seq. 11(1-2):163-6 (2000); canine (Yang, S., supra); and human(Rosnet, O. et al., Blood. 82(4):1110-9 (1993)). Recombinant forms andvariants of FLT-3 ligand are described in, among others, Sudo, Y. etal., Blood 89:3186 (1997) and McClanahan, T. et al., Blood 88:3371-3382(1996)).

Thrombopoietin or “TPO”, also known as megakaryocyte growth anddifferentiation factor (MGDF) or c-Mpl ligand, stimulates theproliferation and differentiation of megakaryocytes, and thus enhancesproduction of platelets in vitro and in vivo (see, e.g., Lok, S. et al.,Stem Cells 12(6):586-98 (1994)), TPO exerts its effects via binding to aspecific cell surface receptor encoded by the proto-oncogene c-mpl. Aswith many other cytokines and growth factors, TPO is characterized bythe presence of an antiparallel four-helix bundle fold (Feese, M. D. etal., Proc. Natl. Acad. Sci. USA. 101 (7):1816-21 (2004)). Amino acid andnucleic acid sequences for thrombopoietin are known for, among others,murine (Lok, S., Nature 369(6481):565-568 (1994)); rat (Ogami, K. etal., Gene 158(2):309-10 (1995)); and human (Foster, D. C. et al., Proc.Natl. Acad. Sci. USA. 91(26):13023-13027 (1994); Bartley, T. D. et al.,Cell 77 (7):1117-1124 (1994)). Recombinant and variant forms of TPO aredescribed in, among others, Souryi, M. et al., Cell 63:1137-1147 (1990);Gurney, A. L. et al., Blood 85(4):981-8 (1995); Wada, T. et al., BiochemBiophys Res Commun. 213(3):1091-8 (1995); Park, H. et al., J Biol Chem.273(1):256-61 (1998); and Jagerschmidt, A. et al., Biochem. J. 333 (Pt3):729-34 (1998).

Erythropoietin or EPO regulates red blood cell production by stimulatingthe expansion and maturation of immature erythrocytes and megakaryocytedevelopment (see, e.g., Fisher, J. W., Proc. Soc. Exp. Biol. Med.216(3):358-69 (1997)). It exerts its effect by binding to the EPOreceptor. Although the primary site for EPO synthesis is the renalcortex of the kidney, lower levels of EPO are synthesized by the liverand macrophages in the bone marrow. EPO is structurally similar to TPO,as characterized by the presence of a four-helix bundle (Feese, M. D. etal., Proc. Natl. Acad. Sci. USA. 101(7):1816-21 (2004)). Amino acid andnucleic acid sequences for EPO are known for, among others, mouse(Shoemaker, C. B. and Mitsock, L. D. et al., Mol Cell Biol. 6(3):849-58(1986)); rat (Nagao, M. et al., Biochim. Biophys. Acta. 1171(1):99-102(1992)); sheep (Fu, P. et al., Mol Cell Endocrinol. 93(2):107-16 (1993);canine (Wen, D. et al., Blood 82(5):1507-16 (1993)); bovine (Suliman, H.B. et al., Gene 171(2):275-80 (1996)); rabbit ((Vilalta A. et al.,Biochem Biophys Res Commun. 284(3):823-7 (2001)); swine (Wen, D. et al.,supra; David, R. B. et al., Domest. Anim. Endocrinol. 20(2):137-47(2001); monkey (Lin, F. K. et al., Gene. 44(2-3):201-9 (1986;)); andhuman (Lin, F. K. et al., Proc. Natl. Acad. Sci. USA 82 (22): 7580-7584(1985); Gasson, J. C. et al., Nature 315(6022):768-71 (1985)).Recombinant forms and variants of EPO are described in Barbone, F. P. etal., Nephrol Dial Transplant. 14 Suppl 2:80-4 (1999); Boissel, J. P. etal., J. Biol. Chem. 268(21):15983-93 (1993). EPO is commerciallyavailable under the trade names Epogen® (Amgen, Thousand Oaks, Calif.,USA), Epogin® (Chugai Pharmaceuticals, JAPAN), Eprex® (Janssen-Cilag,Saunderton, UK), Recormon® (Roche, Basel, Switzerland), and Procrit®(Ortho Biotech., Bridgewater, N.J., USA).

Variants as used herein include substitutions, deletions, insertions ofany amino acid in the cytokine or growth factor sequence, where thevariant retains the biological activity associated with each cytokine orgrowth factor. Substitutions of one or more amino acid residues may bemade while preserving biological activity, and typically involvessubstitution of one amino acid with a homologous amino acid, alsoreferred to herein as “conservative substitution.” In some instances anon-conservative substitutions may also be made. Homologous amino acidsmay be classified based on the size of the side chain and degree ofpolarization, including, small non-polar (e.g., cysteine, proline,alanine, threonine); small polar (e.g., serine, glycine, aspartate,asparagine); intermediate polarity (e.g., tyrosine, histidine,tryptophan); large non-polar (e.g., phenylalanine, methionine, leucine,isoleucine, valine). Homologous amino acid may also be grouped asfollows: uncharged polar R groups (e.g., glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine); acidic amino acids (e.g.,aspartic acid, glutamic acid); and basic amino acids (lysine, arginine,and histidine). Examples of conservative variants include thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine, or methionine for another; the substitution of one polarresidue for another polar residue, such as substitution of one argininefor lysine, glutamic acid for aspartic acid, or glutamine forasparagines; and the substitution of one hydroxylated amino acid serineor threonine for another.

Deletions range from about 1 to about 20 residues, although in somecases, deletions may be much larger, particularly when the cytokine orgrowth factor has physically separable structural and/or functionaldomains. For instance, a variant of FL is the cleaved extracellulardomain, which, as discussed above, retains biological activity whenseparated from the sequences containing the transmembrane and cytoplamicdomains. In addition, amino acids may be added to the amino or carboxyterminus, or in the amino acid sequences joining structural domains,such as a peptide region joining alpha helixes or beta sheets present inthe cytokine or growth factor. Variants for each of the cytokines andgrowth factors will be apparent to the skilled artisan, exemplaryreferences of which are given above.

Cytokine and growth factor combinations are chosen to expand thecommitted myeloid progenitor cells while limiting expansion of HSCs andrestricting the production of terminally differentiated cells of themyeloid lineage. In one embodiment, the mixture of cytokines and growthfactors has the basic composition SCF, FL, and TPO. This cytokine andgrowth factor mixture permits limited expansion HSCs and is permissivefor differentiation of HSCs and other progenitor cells into MPs,including, among others, CMP, GMP, and MEPs.

In a further aspect, the basic composition is supplemented with anadditional cytokine, including IL-3, IL-6, or IL-11, or combinationsthereof. Thus, in one embodiment, the mixture of cytokines and growthfactors has the composition SCF, FL, TPO, and IL-3, a cytokine mixturethat appears to efficiently expand human myeloid progenitor cells. Inanother embodiment, the mixture of cytokines and growth factors has thecomposition SCF, FL, TPO, and IL-6, a cytokine mixture that appears toefficiently expand murine myeloid progenitor cells. Yet in anotherembodiment, the mixture of cytokine and growth factor has thecomposition SCF, FL, TPO, IL-6, and IL-11.

In instances where more mature cells of the myeloid lineage are desiredto enhance immediate protection against neutropenia and/orthrombocytopenia, the cytokines G-CSF or GM-CSF is added to theforegoing cytokine mixtures. This may be done after an initial period ofgrowth in media lacking G-CSF or GM-CSF to permit expansion of moreprimitive myeloid progenitor cells prior to promoting differentiationinto the progenitor cells that are further along in the myeloid lineage.

It is to be understood that different cytokine and growth factormixtures above may be used to favor expansion of specified progenitorcells. Preferential expansion of certain cell populations is desirableif the cells are to be used to treat a single condition or a combinationof conditions (e.g., neutropenia and thromobocytopenia), and theduration of therapeutic effect desired. For instance, presence of CMPswill provide prolonged amelioration of both neutropenia andthrombocytopenia since CMPs are able to differentiate into granulocytes,macrophages, megakaryocytes, and erythroid cells. On the other hand, acell population having a significant proportion of GMPs, as compared toCMPs and MEPs, will provide amelioration of neutropenia but have lesstherapeutic impact on thrombocytopenia since GMPs differentiate intogranulocytes and macrophages but not megakaryocytes. Conversely, a cellpopulation having a significant proportion of MEPs, as compared to CMPsand GMPs, will provide amelioration of thrombocytopenia but have lesstherapeutic impact on neutropenia since MEPs differentiate intoerythroid cells and megakaryocytes but not granulocytes and macrophages.

The amount of cytokines and growth factors in the expansion medium isthe amount sufficient to support expansion of myeloid progenitor cellsto the specified levels in the cell culture. As representativeembodiments, SCF is used at an amount sufficient to support expansion,generally in the amount of at least about 1 to about 1000 ng/ml, andpreferably at about 50 to about 100 ng/ml. FL is used at an amountsufficient to support expansion, generally in the amount of at leastabout 1 to about 1000 ng/ml, and preferably at about 30 to about 100ng/ml. TPO is used at an amount sufficient to support expansion,generally in the amount of at least about 0.5 to about 500 ng/ml, andpreferably at about 5 to about 50 ng/ml. IL-1 is used at an amountsufficient to support expansion, generally in the amount of at leastabout 1 to about 100 ng/ml, and preferably at about 10 to about 50ng/ml. IL-3 is used at an amount sufficient to support expansion,generally in the amount of at least about 1 to about 100 ng/ml, andpreferably at about 10 to about 50 ng/ml. IL-11 is used at an amountsufficient to support expansion, generally in the amount of at leastabout 1 to about 100 ng/ml, and preferably at about 10 to about 50ng/ml. G-CSF is used at an amount sufficient to support expansion,generally in the amount of at least about 1 to about 1000 ng/ml, andpreferably at about 10 to about 100 ng/ml. GM-CSF is used at an amountsufficient to support expansion, generally in the amount of at leastabout 1 to about 100 ng/ml, and preferably at about 10 to about 100ng/ml. When used, EPO is used at an amount sufficient to supportexpansion, generally in the amount of at least about 1 to about 30 U/ml,and preferably at about 3 to about 10 U/ml. As a general guide, themixture of cytokines and growth factors will emphasize growth of myeloidprogenitor cells while limiting the expansion of hematopoietic stemcells.

Expansion of myeloid progenitor cells is carried out in a basal medium,which is supplemented with the mixture of cytokines and growth factorsdescribed above, sufficient to support expansion of myeloid progenitorcells. The basal medium will comprise amino acids, carbon sources (e.g.,pyruvate, glucose, etc.), vitamins, serum proteins (e.g., albumin),inorganic salts, divalent cations, antibiotics, buffers, and otherpreferably defined components that support expansion of myeloidprogenitor cells. Suitable basal mediums include, by way of example andnot limitation, RPMI medium, Iscove's medium, minimum essential medium,Dulbeccos Modified Eagles Medium, and others known in the art (see,e.g., U.S. Pat. No. 6,733,746). Commercially available basal mediumsinclude, by way of example and not limitation, Stemline™ (SigmaAldrich), StemSpan™ (StemCell Technologies, Vancouver, Canada), Stempro™(Life Technologies, Gibco BRL, Gaithersburg, Md., USA) HPGM™ ((Cambrex,Walkersville, Md., USA), QBSF™ (Quality Biological, Gaithersburg, Md.,USA), X-VIVO (Cambrex Corp., Walkersville, Md., USA) and Mesencult™(StemCell Technologies, Vancouver, Canada). The formulations of theseand other mediums will be apparent to the skilled artisan.

The initial population of cells are contacted with the mixture ofcytokines and growth factors in the basal medium, and cultured to expandthe population of myeloid progenitor cells. Expansion is done for fromabout 2 days to about 14 days, preferably from about 4 days to 10 days,more preferably about 4 days to 8 days and/or until the indicated foldexpansion and the characteristic cell populations are obtained.

In one embodiment, the final cell culture preparation is characterizedby a CMP cell population that is expanded at least about 0.5 fold, about1 fold, about 5 fold, about 10 fold, about 20 fold, or preferably atleast about 30 fold. In the final culture, the myeloid cell populationwill comprise CMPs which are at least about 0.5%, at least about 1%, atleast about 2%, at least about 5%, and at least about 10% of the totalcells in the culture.

In another embodiment, the final cell culture preparation ischaracterized by a GMP cell population that is expanded at least about10 fold, about 20 fold, about 40 fold, and preferably at least about 80fold. In the final culture, the myeloid cell population will compriseGMPs which are at least about 10%, at least about 20%, at least about30%, and preferably at least about 50% of total cells in the culture.Thus, in preferred embodiments, the cell populations are expanded topreferentially enrich for GMP cells.

In yet a further embodiment, the final cell culture preparations arecharacterized by a MEP cell population that is expanded at least about0.1 fold, about 1 fold, about 2 fold, about 5 fold, and preferably about10 fold. In the final cultures, the myeloid cell population willcomprise MEPs which are least about 0.5%, about 1%, about 2%, andpreferably at least about 5% of total cells in the culture.

Generally, in the final culture, the expansion of cells with HSCcharacteristics will be limited to less than about 25 fold, preferablyless that about 15 fold, and more preferably less than about 10 fold,and most preferably less than about 5 fold. Generally, the number of HSCcells will be less than the total number of myeloid progenitor cells(i.e., CMP, GMP, and MEP) in culture.

Although less preferred for practical reasons, in some instances, moreundefined culture mediums including feeder cells may be used toapproximate the microenvironment of the bone marrow where hematopoiesisoccurs. Bone marrow stromal cells, endothelial cells, and mesenchymalcells can produce factors that support development and maintenance ofhematopoietic cells in culture, and may be used for the expansion ofmyeloid progenitor cells. The feeder cell cultures may also besupplemented with the mixture of cytokines and growth factors describedabove to promote cell expansion and development of specified myeloidprogenitor cells. Feeder cell based cultures are described in U.S. Pat.No. 5,879,940; Dexter, T. M. et al., J. Cell Physiol. 91:335-344 (1976);Okubo, T. et. al., Cell Structure and Function 25:133-139 (2000);Shapiro, F. et al., J Hematother 5(6):655-62 (1996)); Coutinho, L. H. etal., “Clonal and long term bone marrow cultures using human bonemarrow,” in Haemotology: A Practical Approach, Testa, N. G. andMolineux, G. eds., Oxford University, Oxford, U K (1992); allpublications are incorporated by reference). Typically, mononuclearcells from bone marrow are cultured in suitable medium (e.g., Iscove'sModified Dulbecco's Medium) until a stromal cell layer forms. Thecultures are then irradiated and seeded with initial population of cellsused for expansion.

Another method based on feeder cell cultures is described in Feugier, P.et al., J Hematother Stem Cell Res 11(1):127-38 (2002)). This techniqueuses immortalized bone marrow endothelial cells modified to expresscytokines and growth factors sufficient to support growth of HSCs and/orprogenitor cells. Recombinant expression vectors encoding the specifiedcytokines and growth factors are introduced into the immortalized cellline, and the cells cultured to generate the layer of factor producingendothelial cells. The cells are then irradiated and the culture seededwith cells used for the expansion. Generally, the HSC and progenitorcells are weakly adherent or non-adherent under these cultureconditions, which permits washing of the expanded cells away from theendothelial cells. For the purposes herein, the cytokine and growthfactor genes introduced into the immortalized cells will reflect thecombinations sufficient to support expansion of the committed myeloidprogenitor cells. Accordingly, in one embodiment, the cytokine genes areselected from those encoding IL-1 (i.e., IL-1β), IL-3, IL-6, IL-11,G-CSF, or CM-CSF. Similarly, the growth factor genes are selected fromthose encoding SCF, FL, TPO, and EPO.

In accordance with the above, in one embodiment, expression vectorscomprising genes encoding SCF, FL, and TPO are introduced into thefeeder cells. In a further embodiment, expression vector comprisinggenes coding for an additional cytokine, including IL-3, IL-6, or IL-11,or combinations thereof, are used with the growth factor combination.Thus, in one embodiment, the genes introduced into the feeder cellsencode SCF, FL, TPO, and IL-3. In a further embodiment, the genesintroduced into the cells encode SCF, FL, TPO, and IL-6. In yet afurther embodiment, the genes encode SCF, FL, TPO, IL-6, and IL-11. Thegene sequences are chosen to express cytokine and growth factor formsthat are active on the cells used for expansion, and thus will generallyreflect the origin of the initial cells used for expansion. For example,if the progenitor cells are of human origin, nucleic acid sequencesencoding human forms of the cytokine are used. When the cells forexpansion are of murine origin, nucleic acid sequences encoding murineor other rodent forms of the cytokine are used. Nucleic acid sequencesthat may be used include those encoding recombinant forms or variants asknown in the art. Various other gene combinations sufficient to supportexpansion of myeloid progenitor cells will be apparent to the skilledartisan.

Cells expanded by the methods above are used without furtherpurification, or are isolated into different cell populations by varioustechniques known in the art, such as immunoaffinity chromatography,immunoadsorption, FACS sorting, or other procedures as described above.Preferably, FACS sorting or immunoadsorption is used. For example, aFACS gating strategy has an initial selection for live cells based oncharacteristic forward scatter (cell size) and side scatter (celldensity) parameters, and a second selection for expression of cellmarkers for myeloid progenitor cells or non-myeloid cells (e.g.,Sca-1^(neg) c-kit^(hi)).

The isolated cell populations may comprise isolated committed myeloidprogenitor cells, isolated CMPs, isolated GMPs, isolated MEPs, asdefined herein. In some situations, an isolated non-myeloid cellpopulation is prepared by removal of committed myeloid cells from theexpanded culture. Isolated cells are generally substantially purepopulations of cells, and will typically have at least about 50%,preferably at least about 75-80%, more preferably at least about 85-90%,and most preferably at least about 95% of the indicated cells with thecharacteristic cell marker phenotype and differentiation potential.

6.4 Allogeneic Mixtures of Myeloid Progenitor Cells

As discussed above, to provide sufficient numbers of cells for treatmentpurposes, and to enable the production of a commercially-feasableclinical product, the cell populations are preferably a mixture ofallogeneic myeloid progenitor cells obtained from a plurality ofallogeneic donors. Although there exists the possibility of an immuneresponse against therapeutic cells mismatched with the recipient's MHC,the present therapy is meant to provide temporary protection as opposedto more permanent protection afforded by reconstitution of hematopoiesisby HSCs.

Significantly, as described herein, the possibility of GVHD is greatlyminimized by using isolated committed myeloid progenitor cells that aredepleted of mature lymphoid cells. In one embodiment, this isaccomplished by removal of such cells from the expanded cell populationprior to administration. In an alternative and preferred embodiment,this is accomplished by starting with a substantially pure population ofCD34+CD90+ HSCs.

Mixtures of allogeneic cells include, allogeneic mixtures of myeloidprogenitor cells, mixtures of isolated CMPs, mixtures of isolated GMPs,mixtures of isolated MEPs, or combinations thereof. Cells in the mixturemay be completely matched allogeneic, partially mismatched allogeneic,and/or fully mismatched allogeneic cells with respect to the MHC of thetransplant recipient, and may be from related donors, usually siblingswith the same parental alleles, or unrelated donors. Determining thedegree of MHC mismatch will employ standard tests known and used in theart.

For instance, there are at least six major categories of MHC genes inhumans, identified as being important in transplant biology. HLA-A,HLA-B, HLA-C encode the HLA class I proteins while HLA-DR, HLA-DQ, andHLA-DP encode the HLA class II proteins. Genes within each of thesegroups are highly polymorphic, as reflected in the numerous HLA allelesor variants found in the human population, and differences in thesegroups between individuals is associated with the strength of the immuneresponse against transplanted cells. Standard methods for determiningthe degree of MHC match examine alleles within HLA-B and HLA-DR, orHLA-A, HLA-B and HLA-DR groups. Thus, tests are made of at least 4, andpreferably at least 6 MHC antigens within the two or three HLA groups,respectively.

In serological MHC tests, antibodies directed against each HLA antigentype are reacted with cells from one subject (e.g., donor) to determinethe presence or absence of certain MHC antigens that react with theantibodies. This is compared to the reactivity profile of the othersubject (e.g., recipient). Reaction of the antibody with an MHC antigenis typically determined by incubating the antibody with cells, and thenadding complement to induce cell lysis lymphocytotoxicity testing). Thereaction is examined and graded according to the amount of cells lysedin the reaction (Mickelson, E. and Petersdorf, E. W., Hematopoietic CellTransplantation, Thomas, E. D. et al. eds., pg 28-37, BlackwellScientific, Malden, Mass. (1999). Other cell-based assays include flowcytometry using labeled antibodies or enzyme linked immuno assays(ELISA).

Molecular methods for determining MHC type generally employ syntheticprobes and/or primers to detect specific gene sequences that encode theHLA protein. Synthetic oligonucleotides may be used as hybridizationprobes to detect restriction fragment length polymorphisms associatedwith particular HLA types (Vaughn, R. W., Methods in Molecular Biology:MHC Protocols 210:45-60 (2002)). Alternatively, primers may be used foramplifying the HLA sequences (e.g., by polymerase chain reaction orligation chain reaction), the products of which can be further examinedby direct DNA sequencing, restriction fragment polymorphism analysis(RFLP), or hydridization with a series of sequence specificoligonucleotide primers (SSOP) (Petersdorf, E. W. et al., Blood92(10):3515-20 (1998); Morishima, Y. et al., Blood 99(11):4200-6 (2002);and Middleton, D. and Williams, F., Methods in Molecular Biology: MHCProtocols 210:67-112 (2002)).

While description of “matched allogeneic” or “mismatched allogeneic” isgiven for human MHC, it is to be understood that similar analysis may beconducted for MHCs for various animal species. These include, by way ofexample and not limitation, mouse, rat (Gill, T. J. et al., TransplantProc. 27(2):1495-500 (1995)), cow (Lewin, H. A, et al., Immunol Rev.167:145-58 (1999), canine (Wagner, J. L. et al., J. Hered. 90(1):35-8(1999)), feline (O'Brien, S. J. and Yuhki, N., Immunol Rev. 167:133-44(1999)), swine (Chardon, P. et al., Genet Sel Evol. 32(2):109-28(2000)), horses (Kydd, J. et al., Vet Immunol lmmunopathol. 42(1):3-60(1994), and primates (Heise, E. R. et al., Genetica 73(1-2):53-68(1987)).

Allogeneic mixtures of myeloid progenitor cells may have varying degreesof match in the MHC. Thus, in one embodiment, progenitor cells thatundergo temporary engraftment and differentiation may be isolated from adonor having a higher degree of MHC match with the recipient than cellsintended to provide a more immediate therapeutic benefit. For instance,CMPs may be from a donor having a complete or partial match with the MHCof the recipient, while GMPs and MEPs may be from mismatched donors.Other combinations will be apparent to those skilled in the art.

Allogeneic mixtures of cells may be made in various ways. In oneembodiment, cells are obtained from different donors and mixed prior totheir expansion in culture. In another embodiment, myeloid progenitorcells from different donors are expanded separately and then mixed afterexpansion to generate the mixtures of allogeneic progenitor cells. Inanother aspect, the mixtures of allogeneic cells are prepared fromunexpanded cells by obtaining myeloid progenitor cells from differentdonors and mixing the cells prior to their infusion into the recipient.Whether expanded or non-expanded cells are used, it is shown in theembodiments herein that allogeneic myeloid progenitor cells areeffective in protecting mammalian subjects with compromisedhematopoiesis from potentially lethal neutropenic and/orthrombocytopenic conditions.

6.5 Cryopreserved Myeloid Progenitor Cells

Surprisingly, as demonstrated for the first time herein, the expandedpopulation of cells described herein can be cryopreserved and stored forfuture use and still retain their functionality. As described above, avariety of mediums and protocols for freezing cells are known in theart. Generally the cells are concentrated, suspended in a mediumsupplemented with a cryoprotectant and/or stabilizer, frozen and storedat a temperature of 0° C. or less. In some embodiments the the cells arestored at −70° C. or less e.g., −80° C., or in liquid nitrogen or in thevapor phase of liquid nitrogen. The cells can be stored in anycryoprotectant known in the art. For example, the cryoprotectant can bedimethyl sulfoxide (DMSO) or glycerol. In some embodiments, the freezingmedium comprises DMSO from about 5-10%, 10-90% serum albumin, and 50-90%culture medium. In some embodiments, the cryopreservation medium willcomprise DMSO about 7.5%, about 42.5% serum albumin, and about 50%culture medium. The cells can be stored in any stabilizer known in theart. For example, the stabilizer may be methyl cellulose or serum.

Prior to freezing, the cells may be portioned into several separatecontainers to create a cell bank. The cells may be stored, for example,in a glass or plastic vial or tube or a bag. When the cells are neededfor future use, a portion of the cryopreserved cells (from one or morecontainers) may be selected from the cell bank, thawed and used.

6.6 Treatment

Cells prepared by the methods described herein are used for treatment ofvarious disorders related to deficiencies in hematopoiesis caused bydisease or myeloablative treatments. As used herein, “treatment” refersto therapeutic or prophylactic treatment, or a suppressive measure forthe disease, disorder or undesirable condition. Treatment encompassesadministration of the subject cells in an appropriate form prior to theonset of disease symptoms and/or after clinical manifestations, or othermanifestations of the disease or condition to reduce disease severity,halt disease progression, or eliminate the disease. Prevention of thedisease includes prolonging or delaying the onset of symptoms of thedisorder or disease, preferably in a subject with increasedsusceptibility to the disorder.

Conditions suitable for treatment with the cells described hereininclude neutropenia, a condition characterized by decrease in the amountof circulating neutrophils, and thromobocytopenia, a conditioncharacterized by less than normal levels of platelets in the peripheralblood. Both conditions may be a result of acquired or inheriteddisorder.

Defective hematopoietic stem cell development known to create lowneutrophil numbers include, among others, reticular dysgenesis,Fanconis's anemia, Chediak-Higashi syndrome, and cyclic neutropenia. Forthrombocytopenia, low platelet levels are manifested in, among others,Wiskott-Aldrich Syndrome, thrombocytopenia with absent radii (TAR), andsystemic lupus erythematosus. Acquired forms of neutropenia andthrombocytopenia occur under similar circumstances, such as withhematological malignancies, vitamin deficiency, exposure to ionizingradiation, viral infections (e.g., mononucleosis, CMV, HIV, etc.), andfollowing treatment with various cytotoxic drugs.

For the present purposes, the cells may be used prophylactically toreduce the occurrence of neutropenia and thrombocytopenia, and theirassociated complications, particularly to lessen infection byopportunistic pathogens in patients that have been treated withmyeloablative agents or have undergone HSCT. In the transplant setting,myeloid cells can be used concurrently or subsequent to stem celltransplantation until the recipients own HSCs or transplanted HSCs beginto restore hematopoiesis and raise neutrophil and platelet levelssufficiently. Infusion of myeloid progenitor cells increases the numberof neutrophils and megakaryocytes in the treated subject, therebyproviding temporary but needed protection during the neutropenic orthrombocytopenic period. Use of myeloid progenitor cell populations, asopposed to more differentiated neutrophils and platelets, provides forlonger lasting protection because of the temporary engraftment ofmyeloid progenitors and their differentiation in vivo.

Further, cells comprising a mixture of CMPs, GMPs, and MEPs have thecapacity to generate a broader therapeutic effect than protectionafforded by infusion of any one single cell population. This arises fromthe rapid effect on neutrophil and/or platelet levels from the moredifferentiated progenitors in the cell population while the moreprimitive committed myeloid progenitors engraft and develop over time tosupply the needed neutrophils and megakaryocytes after the moredifferentiated cells have become depleted. Infusion with a cellpopulation comprising a mixture of progenitor cells may be appropriatefor subjects already suffering from neutropenia or thrombocytopenia,while infusion of isolated cell populations may be suitable forprophylaxis in patients where neutrophil or platelet levels have not yetfallen below a critical level. It is to be noted that while treatmentsmay provide a detectable increase in peripheral cell count or ANC, thisincrease is not a reliable indicator of successful, transientengraftment or efficacy. Thus other measures, such as reduced infectionrate and/or increased survival may be used for determining effectivenessof the treatment.

The amount of the cells needed for achieving a therapeutic effect willbe determined empirically in accordance with conventional procedures forthe particular purpose. Generally, for administering the cells fortherapeutic purposes, the cells are given at a pharmacologicallyeffective dose. By “pharmacologically effective amount” or“pharmacologically effective dose” is an amount sufficient to producethe desired physiological effect or amount capable of achieving thedesired result, particularly for treating the disorder or diseasecondition, including reducing or eliminating one or more symptoms ormanifestations of the disorder or disease. As an illustration,administration of cells to a patient suffering from a neutropeniaprovides a therapeutic benefit not only when the underlying condition iseradicated or ameliorated, but also when the patient reports a decreasein the severity or duration of the symptoms associated with the disease.Therapeutic benefit also includes halting or slowing the progression ofthe underlying disease or disorder, regardless of whether improvement isrealized. Pharmacologically effective dose, as defined above, will alsoapply to therapeutic compounds used in combination with the cells, asfurther described below.

Cells for infusion include the expanded cell populations withoutadditional purification, or isolated cell populations having definedcell marker phenotype and characteristic differentiation potential asdescribed herein. Expanded cells may be derived from a single subject,where the cells are autologous or allogeneic to the recipient.Accordingly, in one embodiment, the therapeutic cells comprise isolatedcommitted myeloid progenitors. In other embodiments, the cells compriseisolated CMPs, isolated GMPs, isolated MEPs, or combinations thereof. Inother embodiments, the cells used for the infusion comprise non-myeloidcells prepared as described herein.

It is to be understood that cells isolated directly from a donor subjectwithout expansion in culture may be used for the same therapeuticpurposes as the expanded cells. Preferably, the isolated cells are asubstantially pure population of cells. These unexpanded cells may beautologous, where the cells to be infused are obtained from therecipient, such as before treatment with cytoablative agents. In anotherembodiment, the unexpanded cells are allogeneic to the recipient, wherethe cells have a complete match, or partial or full mismatch with theMHC of the recipient. As described above, the isolated unexpanded cellsare preferably obtained from different donors to provide a mixture ofallogeneic myeloid cells.

Transplantation of cells into an appropriate host is accomplished bymethods generally used in the art. The preferred method ofadministration is intravenous infusion. The number of cells transfusedwill take into consideration factors such as sex, age, weight, the typesof disease or disorder, stage of the disorder, the percentage of thedesired cells in the cell population (e.g., purity of cell population),and the cell number needed to produce a therapeutic benefit. Generally,the numbers of expanded cells infused may be from about 1×10⁴ to about1×10⁵ cells/kg, from about 1×10⁵ to about 10×10⁶ cells/kg, preferablyabout 1×10⁶ cells to about 5×10⁶ cells/kg of body weight, or more asnecessary. In some embodiments, the cells are in a pharmaceuticallyacceptable carrier at about 1×10⁹ to about 5×10⁹ cells. Cells areadministered in one infusion, or through successive infusions over adefined time period sufficient to generate a therapeutic effect.Different populations of cells may be infused when treatment involvessuccessive infusions. A pharmaceutically acceptable carrier, as furtherdescribed below, may be used for infusion of the cells into the patient.These will typically comprise, for example, buffered saline (e.g.,phosphate buffered saline) or unsupplemented basal cell culture medium,or medium as known in the art.

6.7 Adjunctive Treatments

A variety of adjunctive treatments may be used with the cells, expandedor unexpanded, described above. For treating neutropenia and relatedconditions, the expanded cells may be used in combination with otheragents and compounds that enhance the therapeutic effect of the infusedcells or treat complications arising from neutropenia. In one aspect,the adjunctive treatments include, among others, anti-fungal agents,anti-bacterial agents, and anti-viral agents. Use of these agents isalso suitable for thrombocytopenia, either as prophylaxis to reduce theoccurrence of infections or address any ongoing infections that lead todestruction of platelets.

In one aspect, the adjunctively administered agent is an anti-fungalagent. Fungal infections are one of the major causes of mortality inpatients suffering from neutropenia, being a significant problem inpatients who have undergone myeloablative therapy and HSCT. Recipientswith delayed engraftment and patients who develop GVHD typically haveprolonged neutropenia, and thus are at high risk for fungal infections.Types of fungal infections are varied, and include, among others,candidiasis (e.g., with candida krusei, candida glabrata, candidaalbicans, candida tropicalis), aspergillosis (e.g., with aspergillusfumigatus, aspergillus flavus), mucormycosis (e.g., with rhizobiumarrhizus, absidia corymbifera, rhizomucor pusillus), cryptococcosis,histoplasma capsulatum, and coccidioides immitis.

Anti-fungal agents for adjunctive administration will generally be asystemic antifungal agent. One useful antifungal agent of this type isamphotericin B from the family of polyene macrolide antibiotics.Amphotericin B is available in various formulations, including as acomplex with deoxycholate; in a colloidal suspension with cholestearylsulfate; and encapsulated in liposomes made of soy lecithin,cholesterol, and distearoylphosphatidylglycerol. Other formulations areknown in the art.

Another antifungal agent is flucytosine, a fluorinated pyrimidine.Deamination of flucytosine by the fungus generates 5-flurouracil, ananti-metabolite and DNA synthesis inhibitor. Flucytosine is typicallyused for infections of cryptococcus and candiadosis. Although usedalone, flycytosine is generally used in combination with amphotericin B.

Imidazoles and triazoles represent a broad class of azole basedantifungal agents. It is believed that imidazoles and triazoles inhibitsterol 14-α-demethylase, resulting in impaired biosynthesis ofergosterol and disruption of cell membrane based activities, such aselectron transport. Azole based antifungals are effective againstcertain types of candiadosis, such as candida albicans, candidaglabrata, and candida neoformans. Exemplary azole antifungals suitablefor systemic administration include, among others, ketoconzaole,itracanazole, fluconazole, econazole, voriconazole, and tercanozole.

In addition to fungal infections, a patient with neutropenia issusceptible to infection with a variety of bacterial pathogens. Patientsundergoing myeloablative regimens and HSCT have high rates of bacterialinfection with both Gram positive (e.g., streptococcus andstaphylococcus aureus) and Gram negative bacteria (e.g., E. coli. andpseudomonas aeruginosa). Septecemia is a common occurrence. In addition,delayed engraftment and impaired restoration of immune responses againstencapsulated bacteria, such as streptococcus pneumoniae or haemophilusinfluenza, increases the morbidity rate for transplant recipients withGVHD.

Adjunctive antibacterial therapy can use any known antibiotics suitablefor the particular bacterial pathogen. These include both wide spectrumantibiotics and more targeted anti-bacterial compounds. Various classesof anti-bacterial agents suitable with the expanded myeloid cellsinclude, by way of example and not limitation, quinolones andfluoroquinolones, β-lactam antibiotics, aminoglycosides, tetracyclins,macrolides, and various cogeners thereof. Exemplary quinolone compoundsinclude ciprofloxacin, ofloxacin, sparfloxacin, lomefloxacin, andmoxifloxacin. Exemplary β-lactam antibiotics include penicillins (e.g.,penicillin G, penicillin V), ampicillin, carbenicillin, methicillin,carbapenem, and cephalosporins (e.g., cephalothin, cefamandole,cefaclor, cefonicid, cefotetan, cefatoxime, ceftazidime, ceftizoxime,cefepime). Exemplary aminoglycosides include neomycin, streptomycin,kanamycin, gentamicin, tobramycin, amikacin, and netilmicin. Exemplarymacrolides include erythromycin, clarithromycin, and azithromycin. Otherantibiotics will be apparent to the skilled artisan.

Viral infections are also problematic in myeloablated patients andHSCTs. Generally the increased risk of viral infection results fromimpaired cell mediated immunity brought on by the myeloablative therapy.Many of these infections arise from reactivation of latent virusexisting in a seropositive patient or in the cells of a seropositivedonor. Viruses commonly encountered include, among others,cytomegalovirus, herpes simplex virus, varicella zoster virus,herepesvirus-6, Epstein Barr virus; adenoviruses, and the like. As anadjunct to the cell infusions, anti-viral compounds selected are thoseappropriate to the viruses encountered by the patient. Useful antiviralcompounds include, by way of example and not limitation, acyclovir,cidofovir, ganciclovir, idoxuridine, penciclovir, valganciclovir,valacyclovir, vidarabine, amantadine, rimantadine, zanamivir,fomivirsen, imiquimod, and ribavirin. Therapeutics directed againstretroviruses include, among others, nucleoside reverse transcriptatseinhibitors (e.g., zidovudine, didanosine, stavudine, zalcitabine,lamividudine), non-nucleoside reverse transcriptase inhibitors (e.g.,nevirapine, efavirenz, delvirudine), and protease inhibitors (e.g.,saquinivir, indinavir, ritonavir, nelfinavir, amprenavir, andlopinavir).

The antifungal, antibacterial, and antiviral agents may be used asprophylaxis to reduce the occurrence of the infection, or uponappearance of the disease. Prophylaxis is particularly indicated forfungal infections common in immunosuppressed patients, and for viralinfections in seropositive patients or seropositive transplant donors.Accordingly, embodiments for therapeutic purposes include combinationsof the expanded or isolated myeloid progenitor cells and the antifungal,antibacterial, or antiviral compounds.

An additional adjunctive treatment for thrombocytopenia and relatedconditions include transfusions with platelets as a temporary measure torestore platelet count to safe levels. Transfusions are continued untilrecovery of platelet production by transfused cells.

In a further embodiment, the adjunctively administered agent is acytokine or growth factor that enhances differentiation and mobilizationof terminally differentiated myeloid cells, particularly granulocytes,macrophages, megakaryocytes and erythroid cells. For enhancinggranulocyte development, the cytokines C-CSF and GM-CSF may be used.G-CSF is effective in accelerating engraftment and production ofneutrophils in HSCT. In another embodiment, the cytokine or growthfactor is thrombopoietin. Administration of TPO enhances engraftment oftransplanted progenitor cells and promotes development of megakaryocytesand platelets (Fox, N et al., J. Clin. Invest. 110:389-394 (2002);Akahori., H. et al., Stem Cells 14(6):678-689 (1996)).

A variety of vehicles and excipients and routes of administration may beused for adjunctive therapy, as will be apparent to the skilled artisan.Representative formulation technology is taught in, inter alia,Remington: The Science and Practice of Pharmacy, 19th Ed., MackPublishing Co., Easton, Pa. (1995) and Handbook of PharmaceuticalExcipients, 3rd Ed, Kibbe, A. H. ed., Washington D.C., AmericanPharmaceutical Association (2000); hereby incorporated by reference intheir entirety.

The pharmaceutical compositions will generally comprise apharmaceutically acceptable carrier and a pharmacologically effectiveamount of the compounds, or mixture of thereof, or suitable saltsthereof. The pharmaceutical composition may be formulated as powders,granules, solutions, suspensions, aerosols, solids, pills, tablets,capsules, gels, topical crémes, suppositories, transdermal patches, andother formulations known in the art.

As used herein, “pharmaceutically acceptable carrier” comprises any ofstandard pharmaceutically accepted carriers known to those of ordinaryskill in the art in formulating pharmaceutical compositions. Thus, thecompounds, by themselves, such as being present as pharmaceuticallyacceptable salts, or as conjugates, may be prepared as formulations inpharmaceutically acceptable diluents; for example, saline, phosphatebuffer saline (PBS), aqueous ethanol, or solutions of glucose, mannitol,dextran, propylene glycol, oils (e.g., vegetable oils, animal oils,synthetic oils, etc.), microcrystalline cellulose, carboxymethylcellulose, hydroxylpropyl methyl cellulose, magnesium stearate, calciumphosphate, gelatin, polysorbate 80 or the like, or as solid formulationsin appropriate excipients.

The pharmaceutical compositions will often further comprise one or morebuffers (e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants(e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene,butylated hydroxyanisole, etc.), bacteriostats, chelating agents such asEDTA or glutathione, solutes that render the formulation isotonic,hypotonic or weakly hypertonic with the blood of a recipient, suspendingagents, thickening agents, preservatives, flavoring agents, sweeteningagents, and coloring compounds as appropriate.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the compositions, the type of carrier will typicallyvary depending on the mode of administration. The therapeuticcompositions may be formulated for any appropriate manner ofadministration, including for example, oral, nasal, mucosal, rectal,vaginal, topical, intravenous, intraperitoneal, intradermal,subcutaneous, and intramuscular administration.

For parenteral administration, the compositions can be administered asinjectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier thatcan be a sterile liquid such as sterile pyrogen free water, oils,saline, glycerol, polyethylene glycol or ethanol. Additionally,auxiliary substances, such as wetting or emulsifying agents,surfactants, pH buffering substances and the like can be present incompositions. Other components of pharmaceutical compositions are thoseof petroleum, animal, vegetable, or synthetic origin, for example,non-aqueous solutions of peanut oil, soybean oil, corn oil, cottonseedoil, ethyl oleate, and isopropyl myristate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles, as indicated above. Alternatively, apharmaceutical composition may be stored in a freeze-dried conditionrequiring only the addition of a sterile liquid carrier immediatelyprior to use. In a preferred embodiment, a pharmaceutical composition isprovided comprising the subject expanded myeloid progenitor cellscryopreserved in a suitable cryopreservation medium, which can then bethawed and resuspended as needed for administration to a patient.

The amount administered to the host will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis or therapy, the state of the host, the manner ofadministration, the number of administrations, interval betweenadministrations, and the like. These can be determined empirically bythose skilled in the art and may be adjusted for the extent of thetherapeutic response. Factors to consider in determining an appropriatedose include, but is not limited to, size and weight of the subject, theage and sex of the subject, the severity of the symptom, the stage ofthe disease, method of delivery of the agent, half-life of the agents,and efficacy of the agents. Stage of the disease to consider includeswhether the disease is acute or chronic, relapsing or remitting phase,and the progressiveness of the disease.

Determining the dosages and times of administration for atherapeutically effective amount are well within the skill of theordinary person in the art. For example, an initial effective dose canbe estimated from cell culture or other in vitro assays. A dose can thenbe formulated in animal models to generate a circulating concentrationor tissue concentration, including that of the IC₅₀ as determined by thecell culture assays.

In addition, toxicity and therapeutic efficacy are generally determinedby cell culture assays and/or using experimental animals, typically bydetermining a LD₅₀ (lethal dose to 50% of the test population) and ED₅₀(therapeutically effectiveness in 50% of the test population). Guidanceis found in standard reference works, for example, Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, 10th Ed. (Hardman, J. G. etal., eds.) McGraw-Hill, New York, N.Y. (2001).

For the purposes of this invention, the methods of administration arechosen depending on the condition being treated, the form of the subjectantibodies, and the pharmaceutical composition. Administration of thetherapeutic compounds can be done in a variety of ways, including, butnot limited to, subcutaneously, intravenously, intraperitoneally,intramuscularly, and possibly direct injection to specified organs suchas e.g., spleen or bone marrow, although systemic administration ispreferred. Administration of the pharmaceutical compositions may bethrough a single route or concurrently by several routes.

The compositions may be administered once per day, a few or severaltimes per day, or even multiple times per day, depending upon, amongother things, the indication being treated and the judgement of theprescribing physician.

Kits

Other embodiments of the compositions described herein are kitscomprising the expanded and/or isolated myeloid progenitor cells,cytokines and growth factors (e.g., G-CSF, GM-CSF, TPO) and/oradjunctive therapeutic compounds. A label typically accompanies the kit,and includes any writing or recorded material, which may be electronicor computer readable form (e.g., disk, optical disc, memory chip, ortape) providing instructions or other information for use of the kitcontents.

7. EXAMPLES 7.1 Example 1: Experimental Methods

Preparation of HSC cells from mice. For obtaining mouse bone marrowcells, animals are euthanized and the femur/tibia removed and cleaned ofmuscle. The bones are crushed into a pulp using a pestle and mortar, themarrow filtered through a nylon screen, and then centrifuged at 1200 RPMfor 5 minutes. Cells are resuspended in 1 ml ACK solution (0.3M NH₄Cl,0.2M KHCO₃, MiliQ filtered water) for 3-4 minutes on ice, and thenwashed by filling the tube with staining media (HANKs buffered salinecontaining 2% FCS and 2 mM EDTA, w/o calcium, w/o magnesium, w/o phenolred). Cells are centrifuged, filtered, and resuspended in stainingmedia, and mouse IgG (1:50 dilution of a 1 mg/ml stock, Sigma, St LouisMo.) are added. Cells are incubated on ice for 10-15 minutes and thenmixed with CD117 microbeads (Miltenyi Biotech, Auburn Calif.) at avolume of 10 μl/mouse in a final volume of 100 μl/mouse staining media.Cells are incubated on ice for 25 minutes. Cells are washed, resuspendedin staining media at a final volume-1 ml/mouse, and filtered through anylon screen. Cells are enriched using an AutoMACs (Miltenyi, Auburn,Calif.), according to manufacturer's directions using the posseldssetting.

Following enrichment, cells are resupended at about 1×10⁸ cells/ml instaining media and the following directed conjugated antibodies(eBioscience, San Diego, Calif.) are added at the appropriateconcentration: Sca-1 allophycocyanin (APC), c-kitR-phycoerythrin-cyanine 7 tandem (PE-Cy7), Thy-1.1 fluoresceinisothiocyanate (FITC), lineage (CD3, CD4, CD5, CD8, B220, mac-1, Gr-1,and Ter119) R-phycoerythrin (PE).

Cells are incubated on ice for 25 minutes, washed, centrifuged, andresuspended in staining media. Propidium iodide (PI) is added to excludedead cells. Mouse KTLS-HSC,c-kit^(high)Thy^(low)Sca-1^(pos)lineage^(neg) are isolated by FACS.

Cell Culture and Expansion.

Lin^(neg/low) KTLS-HSC are sorted from BA mice (H2kb) and plated in 500ul/well serum-free medium containing the cytokine and growth factorcombination c-KitL, FL, TPO and IL-6 (X-Vivo15 basal medium (CambrexBioscience, MD); penstrep (100×), glutamax (100×), 2-mercaptoethanol(5×10⁻⁵M), c-KitL (50 ng/ml), FL (30 ng/ml), TPO (5 ng/ml), and IL-6(long/ml) (Biosource, Camarillo, Calif. and R & D Systems, Minneapolis,Minn.). The cells are plated at about 10,000 cells/well in 24 wellplates. The cells are cultured for 7 days to obtain MP_(c)(culture-derived MP). Cells are fed with 500 ul/well on day 2, and onday 4 half of the media is replaced with fresh media. On day 5, cellsare transferred to 6 well plates with an addition of 1 ml fresh media.On day 7, the cultured cells are collected and two small aliquots areremoved for analysis. The aliquots are mixed with 30,000 beads andstained to analyze for MP (CMP/GMP/MEP) and HSC content. In addition,cell are stained with trypan blue and counted on a haemocytometer. Theanalysis data provides information for calculating fold expansion andtotal cell numbers of HSC and MP (CMP/GMP/MEP).

Analyzing In Vitro Cultures for Myeloid Cell Types.

A suspension of 6.7 μM beads (Spherotech, Libertyville, Ill.) isprepared by adding 4-5 drops of beads to 1 ml of staining medium (SM).Beads are counted using a hemocytometer and trypan blue (1:10 dilution).Beads are at a stock concentration >2×10⁶ beads/ml and counted dailybefore use when using the suspension over multiple days. Using apipette, 20K beads are added to each well of cells to be analyzed. Beadsuspension is vortexed between each sample prior to adding beads to thewells. If wells are to be split into multiple samples, i.e., 3 samples,appropriate number of beads is added back (i.e., 3×20K beads for normalsample=60K/well), so that each sample will have an endpoint of about 20Kbeads for analysis.

Staining for Mouse HSC in Expanded Cell Populations with IL7R Staining:

Cells are removed from each well, washed and then transferred to acorresponding conical FACS tube. Cells are centrifuged for 5 min @ 1100rpm, and the supernatant removed. 50 ul of blocking antibody (rat IgGand Mouse IgG 1:50) is added, incubated for 10 min, followed by additionof 50-100 ul of CD117-biotin (2× concentration) to each tube. Followingincubation on ice for 20 minutes in the dark, cells are washed with 2-3ml SM, centrifuged, and resuspended in 50-100 μl of the followingantibody solution using appropriate concentrations of antibodies(eBioscience, San Diego, Calif.): Streptavidin Cascade Blue (MolecularProbes, Eugene, Oreg.), Sca-1 allophycocyanin (APC), Thy-1.1 fluoresceinisothiocyanate (FITC), IL-7Rα R-phycoerythrin (PE) and B220, Mac-1, GR-1R-phycoerythrin-cyanine 7 tandem (PE-Cy7). Following incubation on icefor 25 minutes, cell are washed, centrifuged, and resuspended instaining media containing PI. Cells are analyzed for HSC by FACS.

Staining for mHSC in Expanded Cell Populations without IL7R Staining.

Cells are removed from each well, washed and then transferred to acorresponding conical FACS tube. Cells are centrifuged for 5 min @ 1100rpm, and the supernatant removed. 50 ul of blocking antibody (rat IgGand Mouse IgG 1:50) is added followed by 50-100 μl of antibody solutionusing appropriate concentrations of the following antibodies(eBioscience, San Diego, Calif.): Sca-1 allophycocyanin (APC), Thy-1.1fluorescein isothiocyanate (FITC), c-kit phycoerythrin-cyanine 7 tandem(PE-Cy7), B220, Mac-1, GR-1 R-phycoerythrin. Following incubation on icefor 25 minutes, cell are washed, centrifuged, and resuspended instaining media containing PI. Cells are analyzed for HSC by FACS.

Staining for Myeloid Progenitors in Culture Expanded Cell Populations:

Cells are prepared in the same manner as done for staining of HSC cellsdescribed above. After incubation with 50 ul of blocking antibody (ratIgG and Mouse IgG 1:50), 50-100 ul of CD117-biotin (2× concentration) isadded to each tube, followed by 20 minutes on ice in the dark. Cells arewashed with 2-3 ml SM, centrifuged, and then resuspended in 50-100 μl ofantibody solution at appropriate concentrations: Streptavidin CascadeBlue (Molecular Probes, Eugene, Oreg.), Sca-1 allophycocyanin (APC),CD34 fluorescein isothiocyanate (FITC), 2.4G2 (FcγR) R-phycoerythrin,and B220, Mac-1, GR-1 phycoerythrin-cyanine 7 tandem (PE-Cy7).(eBioscience, San Diego, Calif.). Cells are prepped for FACS analysis asdone for HSC analysis.

Staining Culture Expanded Cells for Mature Progenitor Cell Subsets:

Cells are processed as described above. Following incubation withblocking antibody, 50-100 ul of CD3-biotin, CD4-Biotin, and CD8-Biotin(2× concentration) are added to each tube and incubated for 20 minuteson ice in the dark.

Cells are washed with 2-3 ml staining media, centrifuged, andresuspended in 50-100 μl of antibody solution: Streptavidin Cascade Blue(Molecular Probes, Eugene, Oreg.), B220 (FITC), Ter119 R-phycoerythrin(PE) and Mac-1, GR-1 R-phycoerythrin-cyanine 7 tandem (PE-Cy7)(eBioscience, San Diego, Calif.)). Following incubation on ice for 25min, cells are processed for FACS analysis as described previously.

Mouse Myeloid Progenitor Cell Isolation—Lineage Depletion.

Femur and tibia are processed as described above, and the cellsresuspend in 1 ml of staining media. Blocking rat and mouse IgG (1:50)is added, and the mixture incubated on ice for 10-15 minutes. Cells arecounted and brought up to 10⁸ cells/ml in staining media with thefollowing biotinylated antibodies at predetermined dilutions: D3, CD4,CD5, CD8, CD127, Ter119, Thy-1.1, and GR-1. Cells are incubated on icefor 25 minutes, washed, centrifuged, and then resuspended in 40 ul/mousestreptavidin beads (Miltenyi, Auburn, Calif.). Staining media is addedto a final volume on 100 μl/mouse, incubated on ice for 20 minutes,cells washed twice and resuspended at 10⁸ cells/ml in staining media.This is followed by filtration through a nylon mesh. Lineage positivecells are depleted using an AutoMacs (Miltenyi Biotech, Auburn, Calif.)as per manufacturers instructions using sensitive depletion mode. Aftercounting, the cells are resuspended at 1×10⁸ cells/ml in staining mediacontaining the following antibodies at the appropriate concentrations:streptavidin phycoerythrin-cyanine 5 tandem (PE-Cy5), Sca-1allophycocyanin (APC), CD34 fluorescein isothiocyanate (FITC), 2.4G2R-phycoerythrin (PE) and c-kit R-phycoerythrin-cyanine 7 tandem (PE-Cy7)(Pharmingen and eBioscience, San Diego, Calif.). Cell are incubated withantibody for 25 minutes on ice, washed, centrifuged, and resuspended instaining media containing PI. Following FACS sorting strategy is used:CMPs are sorted based onlineage^(neg/lo)C-kit^(pos)Sca-1^(neg)CD34^(pos)2.4 G2^(low); GMPs aresorted based on lineage^(neg/lo)c-kit^(pos)Sca-1^(neg)CD34^(pos)2.4G2^(pos); and MEPs are sorted based onlineage^(neg/lo)c-kit^(pos)Sca-1^(neg)CD34^(low)2.4G2^(low).

Growth and Inoculation of Aspergillus fumigatus Conidia and Fungal LoadAnalysis:

A loop of spores from a frozen spore stock is placed onto the middle ofa Sabourauds dextrose agar (SDA) culture plate, and the plate sealed andincubated at 37° C. for 2-3 days with periodic checking for any sign ofcontamination. After 2-3 days a lawn of black spores forms on the plate.The plate is gently rinsed with 5 ml of PBS containing 0.05% Tween 80,and the plate gently scraped until the spores are dispersed into thesolution. Spore stock is made by filtering the conidia suspensionthrough sterile gauze to remove hyphae. The solution is dark from thespores and can contain up to 10⁸ conidia per ml. Spore stocks are storedat 4° C. To titrate the spores, serial dilutions are made in PBS/Tween80 and plated on SDA plates. Following an overnight incubation, theplates are examined for number of colonies. For long-term storage ofspores, one volume of the harvested stock spores is mixed with a onevolume of 50% glycerol and stored at −80° C.

Injection of the spores into mice is carried out using a conidiasolution containing 1,000 conidia per ml (as titrated on Sabouraudsdextrose agar plates). Working spore solution (100 μl) is injectedintravenously using tail vein injections into the mice of interest,typically 8 days in the prophylactic studies, after lethal irradiationand reconstitution with HSC and/or MP. Following administration, an 100μl aliquot of the remaining spore solution is plated onto a Sabaurouddextrose agar plate and incubated at 37° C. Colonies are counted thenext day to confirm the presence of the required amount of activeconidia in the injection.

Fungal Load Analysis.

Following anesthesia with inhaled isoflurane, Aspergillus is injectedintravenously using the tailvein. Mice are sacrificed and lungsharvested for examination. Lungs are cultured onto Sabourand dextroseagar plates to detect fungal growth.

Screening Reconstituted Mice for Presence of Donor Cells.

Screening of mice transplanted with mHSC's and/or mMP's for donor cellpopulation is done by collecting approximately 10-15 drops of blood in0.5 ml 5 mM EDTA in PBS at room temperature. One ml of 2% dextran-500 inPBS is added, mixed, and incubated at 37° C. for 30-45 min. Most redblood cells will settle. The resulting supernatant is transferred to anew tube, the cells collected by centrifugation (5 min, 1000 rpm), andred blood cells lysed with 1.0 ml of 1×ACK for 5-6 minutes on ice. Thisis followed by a wash and centrifugation for 5 minutes at 1200 rpm. Ifthe pellet is still red, the wash steps are repeated. Cells are blockedwith rat IgG and mouse IgG (1:50 each) in 50 ul/tube for 10 to 15minutes on ice. Biotinylated Mac-1 and GR-1 (eBioscience, San Diego,Calif.) are added at the appropriate concentration, and incubated on icein the dark for 20 minutes. Cells are washed and centrifuged for 5minutes at 1200 rpm. The following antibodies are added at theappropriate concentrations: Streptaviden Cascade Blue (Molecular Probes,Eugene, Oreg.), CD45.1 allophycocyanin (APC), CD45.2 fluoresceinisothiocyanate (FITC), B220 R-phycoerythrin cyanine tandom (PE-Cy7) andCD3, CD4, CD8 R-phycoerythrin (PE) (eBioscience, San Diego, Calif.).Following a 25 minute incubation on ice, cells are washed, centrifuged,and resuspended in SM containing PI. Cells are analyzed by FACS.

Screening Reconstituted Mice for Donor Cells Using H2 Markers.

Approximately 10-15 drops of blood are collected in 0.5 ml 5 mM EDTA inPBS at room temperature. One ml of 2% dextran-500 in PBS (RD is addedand the mixture incubated at 37° C. for 30-45 min. Most red blood cellswill settle. The supernatant is transferred to a new tube, and cellscollected by centrifugation (5 min, 1000 rpm). Red blood cells are lysedwith 1.0 ml of 1×ACK (0.3M NH₄Cl, 0.2M KHCO₃) on ice for 5-6 minutes,followed by a wash and then a centrifugation for 5 minutes at 1200 rpm.If the pellet is still very red, steps 4-5 are repeated. Cells areblocked with rat IgG and mouse IgG (1:50 each) in 50 ul/tube for 10 to15 minutes on ice. Cells are stained for 20 minutes with the followingantibodies: Mac-1 and GR-1 phycoerythrin-cyanine 7 tandem (PE-Cy7), B220allophycocyanin (APC), CD3, CD4 and CD8 biotin (eBioscience, San Diego,Calif.).

Additional antibodies are used to label MHC markers depending on themice pairing used for transplant: H2Kd-PE (Balb/c) and H2Kb-FITC(C57/B6) or H2Db-PE (C57/B6) and H2Dk-Fitc (AKR). Cells are prepared bycentrifuging for 5 minutes at 1200 rpm and then stained withStreptavidin Cascade Blue (Molecular Probes, Eugene, Oreg.). Following a20 minute incubation on ice, cells are washed, centrifuged for 5 minutesat 1200 rpm, and then stained with PI. Cells are analyzed by FACS.

7.2 Example 2. Ex Vivo Expanded Cryopreserved Allogenic MyeloidProgenitors Protect Against Lethal Fungus in Neutropenic Mice

This study examined whether HSC can be expanded into large numbers offunctional myeloid progenitors ex vivo; whether ex vivo expanded myeloidprogenitors protect allogeneic neutropenic mice from lethal funguscomparable to the protection provided by myeloid progenitors sorted fromBM; and shows myeloid progenitors can be cryopreserved without loss ofactivity.

FIG. 1A-1C is an exemplary experimental design. FIG. 1A shows cellpopulations sorted and analyzed, different marker combinations candistinguish HSC and progenitors. CD117⁺, CD90.1^(lo), Lin^(neg/lo) andSca-1⁺ can be used to identify HSC. CD117⁺, Lin^(neg/lo) and Sca-1^(neg)can be used to identify a mixed population of myeloid progenitors. Theindividual subpopulations (CMP, GMP and MEP) can be distinguished bytheir CD16/CD34 profile. FIG. 1B shows derivation of myeloid progenitorsfrom HSC in culture. Culture derived MPc can be used fresh orcryopreserved. FIG. 1C shows the use of myeloid progenitors to protectneutropenic mice from a fungal challenge. Several parameters, such asthe strains used, time of infection and the number of cells used varybetween experiments. Typical experiments use BALB/c hosts and C57BL/KaMP donors.

Mice.

C57BL/6 Ka, Thy-1.1, CD45.2 mice were bred and maintained at theResearch Animal Facility of Stem Cells Inc, Palo Alto, Calif. BALB/cmice were purchased from Charles River Laboratories. Donor mice wereused 6-8 weeks old, recipient mice 8-16 weeks old.

Recipient Mice were Irradiated with a Cs Irradiator.

BALB/c recipients received a total of 9.2Gy given in two doses at least3 hours apart. All mice were maintained on acidified water and wereswitched to water containing antibiotics (106 U/L polymyxin B sulfateand 1.1 g/L neomycin sulfate) for 4 weeks post-irradiation to reduceopportunistic infections.

Fungal Infections.

A clinical isolate of Aspergillus fumigatus, that has been previouslydescribed (BitMansour A, et al., Blood 100, 4660-4667) was used toinfect mice. Briefly, the fungus was plated onto Sabouraud dextrose agar(BD Biosciences, Cockeysville, Md.) and incubated for at least 48 hoursat 37° C. The conidia were harvested by pouring 10 ml PBS+0.05% Tween80on the fungal lawn. After gentle scraping the resulting solution wasfiltered to remove hyphae and the resulting conidia solution wasmaintained at 4° C. Plating of serial dilutions on Sabouraud agar plateswas used to determine the conidia concentration. Mice challenged with A.fumigatus received between 100-200 conidia injected i.v. into thetail-vein in a total of 150 μl saline.

Flow Cytometry.

KTLS HSC were prepared by flushing bone marrow from femurs and tibiae ofmice, followed by ammonium chloride lysis of the red blood cells. Theresulting cell suspension was enriched for CD117⁺ cells using anAutoMacs device and CD117-microbeads (Miltenyi Biotec). The enrichedcells are stained for CD117PE-Cy7 (2B8), CD90.1^(FITC) (HIS51), LinPE(CD3 (145-2C11), CD4 (L3T4), CD5 (53-7.3), CD8 (53-6.7), CD19 (ID3),B220 (RA3-6B2), CD11b (M1/70), Gr-1 (8C5) TER119 (TER119) and Sca-1APC(D7) (EBioscience, San Diego, Calif.). CD117+, CD90.1lo, Lin^(neg/lo)and Sca-1⁺ cells were double sorted (yield sort followed by a puritysort) using a Becton and Dickinson FACSAria. HSC from CD90.2 strains(e.g. BALB/c) are sorted without CD90 staining as KLS cells.

Bone marrow derived myeloid progenitor cells, a mix of CMP, GMP and MEPwere sorted from mouse bone marrow by enriching bone marrow for CD117⁺cells as described above. The cells are stained and CD117+,Lin^(neg/lo), Sca-1^(neg) cells were sorted.

Tissue Culture.

Ten thousand Lin^(neg/lo) KTLS HSC are sorted from mouse bone marrow andplated in 24 well-plates containing 0.5 ml X-vivo15 (Cambrex)supplemented with 1% Penicillin/Streptomycin (Biosource), 1% Glutamax(Invitrogen) and 50 ng/ml c-KitL, 5 ng/ml Tpo, 10 ng/ml IL-6 (Biosource)and 30 ng/ml Flt3L (R&D Systems). All growth factors are mouserecombinant. Media is added every other day. 50% of the medium isreplaced on day 4, at which point the cells are replated into a 6 wellplate. At the time of harvest on day 7 the total culture volume is 2 ml.containing 2-7×10⁶ cells. Cells are analyzed for the presence of matureprogenitor cells as described above, and either used fresh orcryopreserved. Cultured cells were cryopreserved in 7.5% DMSO, 42.5%Fetal Bovine Serum and 50% Xvivo15 medium. Upon thawing the cells werequantitated using a viable cell count (trypan blue) followed by flowcytometric analysis to confirm that the freeze thaw process had notaffected the progenitor profiles, prior to injection into irradiatedmice.

HSC Expanded Ex Vivo into Functional Myeloid Progenitors.

In order to develop a clinical useful therapy it is desirable togenerate myeloid progenitors in large amounts. Highly purified KTLS HSCwere sorted and deposited in serum-free medium (X-vivo15) supplementedwith growth factors that act on HSC. A number of different cytokinecombinations were tested (data not shown). The combination of KitL,Flt3L, Tpo and IL-6 induced rapid proliferation but only slowdifferentiation of the plated HSC, such that over a one week periodtotal cell expansion for C57BL/Ka derived HSC averaged 500-fold. Flowcytometric analysis of the cells at the end of seven days in cultureindicates that a significant proportion of the cells have the surfacephenotype of various myeloid progenitors (CMP, GMP and MEP) as well asHSC (as indicated in FIG. 2B). FIG. 2B shows average expansion numbersas observed for C57BL/Ka cells and similar total expansions wereobserved with HSC from several other strains including AKR, FVB and SJL.Transplantation experiments show that while significant numbers of HSCwere present after 5 days of culture, few functional HSC remained afterC57BL/Ka HSC were cultured for 7 days.

On average the expansion of the various progenitor populations, combinedin the CD117⁺Lin^(neg/lo) gate, averaged about 100-fold over the numberof KTLS-HSC plated under the conditions used. Thus, these cultureconditions resulted in significantly more MP than can be directlypurified from mouse bone marrow. Furthermore, using methylcelluloseplating of single cells, the progenitor populations identified bysurface marker phenotype have the expected lineage differentiationpotential (data not shown)

In addition to the various well-defined myeloid progenitors thesecultures also contain more differentiated cells, including a smallnumber of relatively mature megakaryocytes. However, as can be seen inFIG. 2A, the majority of cells, while showing signs of myeloidcommitment, are not terminally differentiated and many retain the blastcell characteristics of stem and progenitor cells. FIG. 2A showsMay-Grunwald/Giemsa stained cytospins of a day 7 culture, most cells areimmature, many clearly myeloid committed. Low numbers of relativelymature megakaryocytes are present. FIG. 2B show the yield of differenttypes of progenitors, as defined by surface marker profile, from HSCafter 7 days in serum-free culture.

Ex Vivo Expanded Myeloid Progenitors Protect Allogenic Neutropenic Micefrom Invasive Aspergillus.

Bone marrow derived myeloid progenitors have been used to protectneutropenic mice from fungal infection. This example shows that culturederived myeloid progenitors protect allogenic neutropenic mice frominvasive fungal infections similar to bone marrow derived myeloidprogenitors.

Reconstitution was performed in lethally irradiated mice (BALB/c,CD90.2, CD45.2, H-2^(d)) with 200 syngeneic BALB/c HSC (CD117⁺,Lin^(neg/lo), Sca-1^(neg), KLS) and either 8×10⁴ bone marrow MP sortedas CD117⁺, Lin^(neg/lo), Sca-1^(neg) cells from C57BL/Ka, H-2b bonemarrow or 5×10⁵ Day 7 culture derived MPc cells. The mice were infected,by tail vein injection, with 150 conidia of a clinical isolate ofAspergillus fumigatus 7 days after the initial irradiation and HSC/MPtransfusion. The experiment was repeated three times with 15 mice pergroup (data not shown). As shown in the pooled data in FIG. 3, only 2/15irradiation controls survived for more than 30 days, confirming that thelethal irradiation dose used is relatively low. Injection of 200 KLS HSCat day 0 (HSC-rescue group) fully rescues the irradiated mice; however,only 17/44 of these mice survive injection of 150 A. fumigatus conidia.In contrast, 34/45 mice (76%) that received BM MP and 32/45 mice (71%)that received culture-derived cells survived the fungal challenge andthe culture-derived cells prevent death. Statistical analysis (log rank)show significant protection by both BM MP cells (p<0.0001) andculture-derived cells (p=0.0014) when compared to the stem cells onlygroup. There is no discernable difference in the 30 day survival betweenthe groups that received BM MP cells when and the group that receivedculture derived-cells (p=0.5164). Thus, ex vivo expanded myeloidprogenitors protect neutropenic mice from invasive aspergillosis as wellas bone marrow derived myeloid progenitors.

Cryopreserved Ex Vivo Expanded Allogenic Myeloid Progenitors ProtectAgainst Aspergillosis in Neutropenic Mice.

An advantage of the use of myeloid progenitors, in contrast to maturegranulocyte infusions, is the ability to cryopreserve progenitors cellsprior to use. The frozen MP cells could be stockpiled and thawed whenneeded.

This study is adapted from the standard Fungus experiments with murineMPc. The standard design is to culture C57BL/Ka HSC for 7 days inXvivo15 supplemented with KitL, Flt3L, Tpo and IL-6. On day 0 the hostmice (BALB/c) are lethally irradiated (2×4.4Gy, 4 hours apart) andinjected on the same day with 5×10⁵ cells from the MPc cultures and 200BALB/c HSC (KLS). On day 7 the mice are injected i.v. with 150 conidiaof Aspergillus fumigatus. The mice are inspected daily and the 30 daysurvival is determined.

The experiments described here will differ in that (i) MPc are culturedat several different occasions (ii) the earlier cultures were frozen andstored in liquid nitrogen (iii) fresh and frozen MPc are compared (iv)composition of MPc were analyzed by flow cytometry before freezing andafter thawing.

Mouse cells can be frozen in a cryopreservation media that is a serumfree or a serum containing mix. For all the research experiments used aserum containing cyropreservation media. Serum containing mix: (150 mltotal) with 37.5 ml serum and 22.5 ml DMSO.

-   -   1. Pellet cells    -   2. Resuspend cells in serum free media (IMDM or Xvivo)    -   3. Prepare a metal bowl with ice and water    -   4. Place the tube of resuspended cells in the icy water and        slowly drop an equal volume of the cyropreservation mix from        above to the resuspended cells while gently mixing the tube.    -   5. Pipet the mixture in vial and put freezing apparatus at −80        overnight    -   6. Transfer vial to −180 for long storage.

For thawing cultured mouse cells:

-   -   1. Thaw the vial in 37 C bath until the content is mostly thawed        Resuspend cells in serum free media (IMDM or Xvivo)    -   2. Pipet cells slowly in vial containing DNAse, take out small        aliquot to do initial vial cell count.    -   3. Dropwise add 10 ml of Media (IMDM/DMEM etc with 10% NCS) to        cells while gently rocking the tube to allow slow mixing of        media and cells.    -   4. Spin down cells and resuspend in staining media (HBSS/2% NCS)    -   5. Count

Dec. 1, 2004 Day −14. Sort BS.BA HSC to initiate MPc cultures (frozenMPc). Dec. 8, 2004 Day −7. Harvest MPc, analyze by flow and freeze thecells (plate CD117+ cells in MCM/Terasaki plate). Dec. 9, 2004 SortBS.BA HSC to initiate MPc cultures (fresh). Dec. 16, 2004 Day 0. SortBALB/c HSC, harvest MPc cultures, irradiate 65 BALB/c mice and inject 60BALB/c mice with MPc (fresh or frozen) and/or HSC. Dec. 23, 2004 Day 7.Inject 50 reconstituted mice with 150 conidia of Aspergillus fumigatusDay 5 to 30. Daily records of surviving and dying mice 2005 Test micefor level of reconstitution starting at 4 weeks

Growth Fungus Group n HSC MPc factor (conidia) Day G1: fresh 15 200Fresh, 500,000 No 150 7 MPc G2: frozen 15 200 Frozen 500,000 No 150 711/29 MPc G3: frozen 15 200 Frozen 500,000 No 150 7 12/8 MPc G4: no MP10 200 0 No 150 7 G5: 5 0 No 7 Radiation Ctrl Totals 60 11,000 3 *7,500,000 None 8,250

MP-Cultures.

CD117⁺CD90.1^(lo) Lin^(neg/low)Sca-1⁺ HSC were sorted fromCD117-enriched BS. BA bone marrow as described below. Mouse HSCIsolation-directly conjugated c-kit microbeads. Collect femurs andtibia, clean of muscle. Crush using pestle and mortar. Filter throughnylon screen, 5 mice/tube (10 leg bones and 10 arms). Centrifuge 1200RPM for 5 minutes. Resuspend in 1 ml ACK ˜3-4 minutes on ice. Wash byfilling tube with staining media. Centrifuge. Count cells. Resuspend instaining media ˜50-60 μl mouse, filter, wash tip and filter with another40-50 μl, add rat IgG (1:50)+mouse IgG (1:50) for 10-15 minutes. Remove10 ul whole BM for staining. Add 10 μl/mouse of anti c-kit microbeads(CD117) per mouse, (lot#5040428046). Note: If humerus are added, use 12μll beads/mouse. Incubate on ice 25 minutes. Wash 2×. Resuspend cellsand filter through nylon screen final volume-0.5-1.0 ml/mouse, wash tipand filter mesh with another 0.5 ml. Enrich cells on AutoMACs, useposselds program. Alternatively, prepare Midi column by washing with 3-4ml staining media. Filter cells through nylon mesh to apply to column.Pass cells over column 3× (no more than 10 mice/column). Wash columnwith 5-10 ml staining media. Remove column from magnet and flush cellsfrom column 2×. (note: 10 mice=midi and 5 mice=mini) Count, centrifuge.Resuspend cells at 1×10⁸ cells/ml in staining media plus antibodies.

Antibody Lot # Titer c-kit (2B8) biotin E000225 1:400 Orc-kit (2B8)PE-Cy7 E009158 1:400 Sca-1 APC E007871 1:200 Thy-1.1 FITC E008124 1:400Lineage PE: Ter 119 E005015 1:400 CD3 E009288 1:100 CD5 E004526 1:1600CD8 E009271 1:200 B220 E007026 1:800 CD4 E008789 1:3200 Mac-1 E0058581:6400 GR-1 E008723 1:3200

Incubate on ice for 25 minutes, wash and centrifuge. If c-kit bio usedresuspend in SM (1×10⁸ cells/ml). Stain with streptavidin Cy7-PE (Lot #E006330) 1:800 25 minutes on ice. Wash, centrifuge, resuspend instaining media+PI (1:1000) filter before FACS. Set up comp tubes basedin what colors are used:

Antibody Lot # Titer B220-PE Cy7 E009142 1:400 B220-Bio E004692 1:800Cascade-Blue 65A1-1 1:400 B220-PE E007026 1:400 B220-FITC E005965 1:200B220-APC E011511 1:200 B220-Cy5PE E004592 1:800 PI 1:1000 No stain

Stain whole BM: Centrifuge cells. Resuspend in 100 ul SM+antibodies (seeabove). Incubate on ice 25 minutes. Wash, centrifuge. (If 2B8-biotin isused, resuspend in 100 ul (10⁷ cells/100 μl) and stain with streptavidinCy7-PE 1:800 25 minutes on ice). Wash, centrifuge, resuspend in SM+PI(1:1000), filter before FACS.

Approximately 3×10⁴ HSC were used to initiate MPc cultures. The cellswere cultured in Xvivo15+2ME+Pen/Strep+50 ng/ml KitL+30 ng/mlFlt3L+5ng/ml Tpo+10 ng/ml IL-6 for 7 days as described below.

Large Scale Mouse HSC to Myeloid Progenitor Culture:

Lin^(neg/low) KTLS-HSC are sorted from mouse bone marrow and plated in500 μl/well Xvivo15 supplemented with Pen/Strep, Glutamax andβ-mercaptoethanol as well as KitL, Flt-3L, Tpo and IL-6. The cells arecultured for 7 days to obtain MP_(c) (culture-derived MP). The expectedexpansion during this period for C57BL/Ka-cells is 200-700 fold totalexpansion, with 10-35% of the cells falling in the CD117⁺Lin⁻ gate.Cells are sorted twice, first on yield then followed by a final sort onpurity. For a 100,000 bulk sort, first sort should yield around 300,000cells. In a 24 well plate, 10,000 KTLS-HSC are sorted directly per wellin 500 μl media. Sterile water is added to the outer wells of the plateto prevent evaporation. The cells are incubated at 37° C., 5% CO₂ in afully humidified incubator. On day 2, 500 μl of fresh media is added toeach well. On day 4, half of the media (500 μl) is discarded (pipetcarefully from the top to avoid removing cells). The cells areresuspended (pipet up and down using a P1000) and transferred to a wellon a 6 well plate containing 500 μl fresh medium. The empty well on the24 well plate is rinsed with 500 μl fresh medium, transfer this to thesame well on the 6 well plate. Total volume should be 1.5 ml per well.Some cells will remain attached to the bottom of the 24 well plate,these are discarded. On day 6, add 500 μl of fresh medium. On day 7, thecultured cells are collected and analyzed. In addition, cell are stainedwith trypan blue and counted using a hemocytometer. The analysis dataprovides information for calculating fold expansion and total cellnumbers of HSC and MP (CMP/GMP/MEP).

Reagents for Large scale mouse HSC to Myeloid Progenitor culture:X-vivo15(Cambrex Bio science 04-744Q) supplemented withPenicillin/Streptomycin (100×) Biosource International Inc., Glutamax(100×) Invitrogen, β-Mercaptoethanol (1000×) Sigma Aldrich Fluka Inc.The growth factors include:

Manufacturer Catalog No Stock solution Use at: rmKitL Biosource PMC211525 ng/μl 50 ng/ml rmFlt3L R&D 427-FL 25 ng/μl 30 ng/ml rmTpo BiosourcePMC1144 10 ng/μl  5 ng/ml rmIL-6 Biosource PMC0066 10 ng/μl 10 ng/ml

Culture media for 100,000 cells (500 ul/well) is 5 ml complete Xvivo, 5μllL6, 2.5 μl TPO, 10 μl KilL, 6 μl Flt3L. For 100 ml Complete Xvivo use100/μl β-mercaptoethanol, 1 ML Pen/Strep, 1 ML Glutamax.

After 7 days in culture the cells were harvested and counted. Aliquotswere analyzed as described below. Analyze of HSC/MP by adding 4-5 ofspherotck (6.7 uM) beads to 1 ml of staining medium (SM). Dilute beads1:10 in trypan blue, count using a haemocytometer. Beads should be at astock concentration >2×10⁶ beads/ml. Count beads before each analysis.Add 30,000 beads per sample. Transfer a small aliquot of cells toanalysis tubes. Block with rIgG (1:50) and mIgG (1:50) on ice for 10min. To MP tubes add CD117-BIOTIN @ 1:200. Incubate for 20 min on ice indark. Prepare HSC (Ckit cy7-pE at 1:400, Sca-1 APC at 1:200, Thy-1.1FITCat 1:200, B220 PE at 1:800, Mac-1 PE at 1:800, and GR-1 PE at 1:800) andMP (SA-cascade blue at 1:400, Sca-1 APC at 1:200, CD34 FITC at 1:25,2.4G2 at 1:50, B220 Cy7-PE at 1:800, GR-1 Cy7-PE at 1:800) antibody mix.Wash all tubes w/2 ml sm, spin @ 1100 rpm for 5 min. Incubate withantibody mix for 20 min on ice in dark. Wash cells w/sm and spin.Rresuspend cells in PI media. [PI media (1:1000) 10 ml stock: 10 μl PIin 10 ml sm].

Briefly, flow cytometric analysis was performed to determine thepresence of HSC, CMP, GMP, MEP and more mature cells (CD11b⁺, Gr-1⁺,Ter119⁺). Cytospins (to be stained with May-Grünwald/Giemsa stain) weremade. Approximately 3 times 7.5×10⁷ culture derived cells are needed forinjections, fresh (1×) and frozen (2×).

Freezing of MPc.

MPc were culture-derived from HSC as described. The cells were analyzedby flow cytometry prior to freezing. In addition, single CD117⁺Lin⁻cells were plated, either in Terasaki plates with MPc medium(Xvivo+KitL, Flt3L, Tpo and IL-6) above. The percentage of cells formingcolonies will be determined one week after plating. The cells werefrozen according to protocol as described below. Cells can be frozen ina cryopreservation media that is a serum free or a serum containing mix.For all the research experiments serum containing cyropreservation mediawas used. Serum containing mix: (150 ml total) 37.5 ml serum, 90 mlhetastarch, 22.5 ml DMSO. Pellet cells and resuspend cells in serum freemedia (IMDM or Xvivo). Prepare a metal bowl with ice and water. Placethe tube of resuspended cells in the icy water and slowly drop an equalvolume of the cyropreservation mix from above to the resuspended cellswhile gently mixing the tube. Pipet the mixture in vial and put freezingapparatus at −80 overnight transfer vial to −180 for long storage.

Thawing of Frozen MPc.

Frozen MPc were thawed as described below. Thaw the vial in 37° C. bathuntil the content is mostly thawed Resuspend cells in serum free media(IMDM or Xvivo). Pipet cells slowly in vial containing DNAse, take outsmall aliquot to do initial vial cell count. Dropwise add 10 ml of Media(IMDM/DMEM etc with 10% NCS) to cells while gently rocking the tube toallow slow mixing of media and cells. Add media at approximately 1ml/minute. Spin down cells and resuspend in staining media (HBSS/2%NCS). Then “rest” cells by leaving them at RT for ½ hour. Count/stain orplate cells accordingly.

In this experiment, following thawing the cells were rested forapproximately 1 hour and viability determined by (i) trypanblue/haemocytometer count (ii) PI exclusion flow cytometry analysis. Inaddition, single CD117⁺Lin⁻ MPc were plated and cultured.

Balb/c HSC Sort.

HSC, CD117⁺Lin^(neg/low)Sca-1^(pos), were sorted from 5 BALB/c miceusing as described above. 10,000 HSC (200 per recipient) were needed.The sorted BALB/c HSC were mixed with culture-derived BS. BA MPc in thedesired ratio's and used for injection into lethally irradiated BALB/cmice.

Fungus Injections.

On day 7 the mice will be injected in the tail vein with 150 conidia ofAspergillus fumigatus as described below including the testing of partof the injection solution on Sabauroud-dextrose agar to quantify thenumber of live conidia injected. Aspergillus injection determine theconcentration of conidia for each experiment prior to injections. Totalvolume of 150 μl (sterile 1×PBS+0.05% tween+conidia) is injected permouse. During injections, keep conidia solution on ice and vortex beforeeach syringe filling. Plate 150 μl from the syringe onto a SDA plate andincubate at 37° C. Next day count colonies to confirm the presence ofconidia injected.

To demonstrate that culture-derived myeloid progenitors retain theirability to prevent invasive aspergillosis in neutropenic mice HSC wereplated and cultured as described above. After seven days the cultureswere harvested and analyzed by flow cytometry. The cells were thencryopreserved as described in the above and stored in the vapor phase ofliquid nitrogen. After at least seven days the vials were rapidly thawedin a 37° C. water bath, washed twice with medium. An aliquot was removedfor flow cytometric analysis and the remainder of the cells was used toinject into lethally irradiated allogeneic hosts. FIG. 4A comparessurvival data for fresh MP with that of culture derived myeloidprogenitors that were cryopreserved prior to use, the 30 day survival ofmice challenged with A. fumigatus conidia 7 days after lethalirradiation and reconstitution with 200 syngeneic HSC and 500,000allogeneic culture-derived MP, used either fresh or aftercryopreservation. Survival after fungal challenge in 4 groups of 15 micethat received 500,000 fresh cells differs significantly from that ofmice receiving only HSC (p=0.009, t-test). The same is true for 3 groupsof 15 mice that received 500,000 culture-derived myeloid progenitorsthat had been cryopreserved compared to HSC-only groups (p=0.0329).There is no difference in survival between the groups receiving fresh orfrozen myeloid progenitors (p=0.7205) or the HSC-only groups in bothcases (p=0.5058). FIG. 4B is a comparison of the CD117 Lin stainingprofile of the cultured MP cells before freezing and after thawing, andshows that a freeze/thaw cycle does not affect the CD117 and Linstaining profiles. Similar results were obtained in analysis for themarkers (data not shown).

Reconstitution with Myeloid Progenitors Typically Results in Short-TermEngraftment.

FIG. 5A-5C shows reconstitution with syngeneic MPc illustratesengraftment levels in peripheral blood at 4 weeks post-reconstitutionwith C57BL/Ka cells cultured either 5 days (2.25×10⁵ cells/mouse), 7days (8×10⁵ cells per mouse), or sorted directly from mouse bone marrowas myeloid progenitors (4×10⁴ cells/mouse). As shown in FIG. 5A-5C, few(five percentages) MP-derived cells are present in circulation one monthafter administration. MP-derived cell numbers disappear over time, andare lower at 8 weeks than at 4 weeks post administration (data notshown). The cells that are present are mostly B-lymphocytes. HSC thathave been cultured for five days under MP-inducing conditions show adramatically different pattern of reconstitution. At 4 weeks many of thecirculating cells are MP-derived, and these contain cells representingthe three major lineages, myeloid cells, B cells and T cells. Thissuggests that engraftment is by residual functional HSC. MP-derivedcells numbers are significantly lower 8 weeks post-reconstitution,indicating that the engrafting HSC are mainly restricted to ST-HSCAdministration of cells that have been cultured for eight days displayan engraftment pattern very much like the purified myeloid progenitors.Few MP-derived cells are seen and these mainly represent long-lived Bcells. Thus, the number and type of donor-derived cells was related tothe duration of culture and seven days of culture results in fewMP-derived cells being detected in the host 8 weeks after infusion.

FIG. 6 shows dose response of protection by culture-derived myeloidprogenitors. Combined data from ten independent experiments. Doses areeither plotted as total cell numbers (FIG. 6A) or CD117+Lin− cells inthe MPc (FIG. 6B). These experiments were performed as described forFIG. 5A-5C. BALB/c mice were lethally irradiated and injected withBALB/c HSC and C57BL/Ka MPc, followed by challenge with A. fumigatusconidia at D+7.

Cultured Myeloid Progenitors Rapidly Provide Effective Protection.

FIG. 7 shows combines survival data from 15 separate experiments inwhich fungus was injected at different times after reconstitution of thelethally irradiated mice with 200 syngeneic HSC and 250,000 or 500,000culture-derived MP. FIG. 7 shows recipients mice were able to survivefungal challenge during the first two days following irradiation; byD+3, mice were fully susceptible to infectious challenge.

Mixed Allogenic Cultured Myeloid Progenitors.

The purpose of the experiment was to determine whether mixed allogeneicMPc (i) protects to the same extend as single donor MPc (ii) does notadversely affects the mice. The experiment shows MPc in pooled batchesand cryopreserved as aliquots are a clinically relevant therapeutic.

This study was modified from the standard Fungus experiments with murineMPc. The standard design is to culture C57BL/Ka HSC for 7 days inXvivo15 supplemented with KitL, Flt3L, Tpo and IL-6. On day 0 the hostmice (BALB/c) are lethally irradiated (2×4.4Gy, 4 hours apart) andinjected on the same day with 5×10⁵ cells from the MPc cultures and 200BALB/c HSC (KLS). On day 7 the mice are injected i.v. with 150 conidiaof Aspergillus fumigatus. The mice are inspected daily and the 30 daysurvival is determined.

The experiments described herein differ in that (i) HSC were sorted andcultured from BS. BA, AKR, FVB and SJL mice (ii) reduced numbers of MPcwere given (iii) the fungus was given more rapid post-irradiation.

Jan. 6, 2005 Day −7. Sort BS.BA, AKR, FVB and SJL HSC to initiate 4separate MPc cultures. Jan. 13, 2005 Day 0. Sort BALB/c HSC, harvest MPccultures, irradiate 51 BALB/c mice and inject 45 BALB/c mice with MPcand/or HSC. Jan. 20, 2005 Day 7. Inject 45 reconstituted mice with 200conidia of Aspergillus fumigatus Jan. 21, 2004 to Day 8 to 30. Dailyrecords of surviving and dying Feb. 13, 2005 mice 2005 Test mice forlevel of reconstitution starting at 4 weeks

BS.BA Group n HSC MPc AKR/SJL/FVB Fungus Day HSC noMP 15 200 0 no 200 7conidia BS.BA MPc 15 200 500,000 no 200 7 conidia Mixed MPc 15 200125,000   125,000 each 200 7 conidia Radiation 5 0 0 no 0 n/a Ctrl Total50 9,000 9,375,000 1,875,000 each 9,000

Hosts: BALB/c, H-2^(d), CD90.2, CD45.2 (Charles Rivers Laboratories).Approximately 56 mice necessary, 51 as hosts and 5 as HSC-donors. MPc:C57BL/Ka, H-2^(b), CD90.1, CD45.1 (BS. BA, bred in house); MPc:AKR,H-2^(k), CD90.1, CD45.2 (CRL); MPc:SJL, H-2^(s), CD90.2, CD45.1 (CRL);MPc:FVB, H-2^(q), CD90.1, CD45.2 (CRL). Approximately 5 to 10 mice ofeach strain are necessary, sufficient to generate 8-10×10⁴ HSC. More BS.BA cells are needed than the other strains.

MP-Cultures.

CD117⁺CD90.1^(low) Lin^(neg/low)Sca-1⁺ HSC were sorted fromCD117-enriched BS. BA, AKR and FVB bone marrow as described above. SJLHSC were sorted as CD117⁺Lin^(neg/low)Sca-1⁺ cells. Approximately 3×10⁴HSC will be used to initiate MPc cultures. The cells were cultured inXvivo15+2ME+Pen/Strep+50 ng/ml KitL+30 ng/mlFlt3L+5 ng/ml Tpo+10 ng/mlIL-6 for 7 days as described above After 7 days in culture the cellswere harvested and counted. Aliquots were analyzed. Briefly, flowcytometric analysis was performed to determine the presence of HSC, CMP,GMP, MEP and more mature cells (CD11b⁺, Gr-1⁺, Ter119⁺). Cytospins(stained with May-Grünwald/Giemsa stain) were made. Approximately1.5×10⁷ culture derived cells were needed for injections. Excess cellswere cryopreserved for future use.

Balb/c HSC Sort.

HSC, CD117⁺Lin^(neg/low)Sca-1^(pos), were sorted from 5 BALB/c mice.10,000 HSC (200 per recipient) were needed. The sorted BALB/c HSC weremixed with culture-derived BS. BA MPc in the desired ratio's and usedfor injection into lethally irradiated BALB/c mice.

Fungus Injections.

The mice were injected in the tail vein with 150 conidia of Aspergillusfumigatus as described above. The injections will differ in that thesemice will receive fungus on DAY 4 rather then the usual day 7. Otherprocedures, including the testing of part of the injection solution onSabauroud-dextrose agar to quantify the number of live conidia injected,remain the same.

FIG. 8 shows protection of neutropenic mice by mixed allogeneicculture-derived MP. The results demonstrate the effectiveness of themixed allogeneic MP cells. The experiment used cells grown from 4different strains, all mismatched at major and minor antigens. Mixedallogeneic frozen/thawed MP cells from 3 strains (C57BL/Ka, AKR and FVB)and at half the cell dose resulted in protection without long termreconstitution (data not shown).

Radioprotective Ability of Mismatched Allogenic Cultured DerivedProgenitors.

This experiment used AKR MPc donor and C57/B6Ka recipient. MP wereeither sorted directly from mouse bone marrow or derived in culture fromsorted HSC. Cultured MP were derived over 7 days of culture in X-Vivomedia containing KitL, Flt3L, TPO and IL-6. Following seven days ofculture, cells were analyzed by FACS to determine the frequency of c-kitpositive progenitor cells. A dose of cultured cells was transplantedthat contained 200,000 or 500,000 c-kit positive lineage negativeprogenitors. FIG. 9A shows 30 day radioprotection data from lethallyirradiated mice transplanted with completely MHC mismatched allogeneicMP. Surviving mice have little detectable donor chimerism (FIG. 9B)

Comparison of Radioprotective Ability of Fresh and Frozen MismatchedAllogeneic Cultured Derived Progenitors.

This experiment used AKR MPc donor and C57/B6Ka recipient. MP werederived in culture from sorted HSC. Cultured MP were derived over 7 daysof culture in X-Vivo media containing KitL, Flt3L, TPO and IL-6.Following seven days of culture, cells were harvested and analyzed byFACS to determine the frequency of c-kit positive progenitor cells.Cells were either directly injected into lethally irradiated mice orfrozen and thawed prior to injection. A dose of cultured cells wastransplanted that contained 200,000 c-kit positive lineage negativeprogenitors. FIG. 10 shows 30 day radioprotection data from lethallyirradiated mice transplanted with completely MHC mismatched allogeneicMP. Cyropreserved MPs protect equivalently to MPs that are given at thetime of harvest.

7.3 Example 3. Myeloid Progenitors Initiated from Human HematopoieticStem Cells in Flasks and Bags

Human hpHSC (CD34+CD90+ cells derived from Mobilized Peripheral Blood(MPB) were obtained from healthy volunteers. MPB is enriched for CD34+cells using a Baxter Isolex device. CD34-enriched cells are furtherstained and sorted using a modified Dakocytomation MoFlo of a regular BDFACSaria to obtain CD34+CD90+ cells (“hpHSC”) Cells are used eitherfresh or after cryopreservation, either following the IsolexCD34-enrichment or after CD34+CD90+ sorting on the MoFlo. Samples arefrozen manual or using a step rate freezer.

Freezing of Human Cells.

Cells can be frozen in a cryopreservation media that is a serum free ora serum containing mix. For all the research experiments we are usingserum containing cyropreservation media. Serum containing mix: (150 mltotal). 37.5 ml serum, 90 ml hetastarch, 22.5 ml DMSO. Pellet cells andresuspend cells in serum free media (IMDM or Xvivo). Prepare a metalbowl with ice and water. Place the tube of resuspended cells in the icywater and slowly drop an equal volume of the cyropreservation mix fromabove to the resuspended cells while gently mixing the tube. Pipet themixture in vial and put freezing apparatus at −80 overnight and transfervial to −180 for long storage.

Thawing of Human Cells.

Thaw the vial in 37 C bath until the content is mostly thawed Resuspendcells in serum free media (IMDM or Xvivo). Pipet cells slowly in vialcontaining DNAse, take out small aliquot to do initial vial cell count.Dropwise add 10 ml of Media (IMDM/DMEM etc with 10% NCS) to cells whilegently rocking the tube to allow slow mixing of media and cells. Addmedia at approximately 1 ml/minute. Spin down cells and resuspend instaining media (HBSS/2% NCS) “rest” cells by leaving them at RT for ½hour. Count/Stain or plate cells accordingly

hpHSC are cultured in wells, flasks or bags in Xvivo15+1%Penicillin/Streptomycin, 1% Glutamax and 100 ng/ml KITL, 100 ng/mlFLT3L, 50 ng/ml TPO and 10 ng/ml IL-3, unless indicated otherwise. Thecytokines base mix is rhKITL 100 ng/ml(Amgen) stock: 100 pg/ml; rhTPO 50ng/ml (Biosource) stock: 10 pg/ml; rhFLT3L 100 ng/ml(Amgen)stock: 100pg/ml; rhIL-3 10 ng/ml(Biosource) stock: 10 μg/ml. In some experiments,the additive effect of the following cytokines was tested rhIL-6 10ng/ml, rhIL-11 10 ng/ml, rhGM-CSF 10 ng/ml, rhG-CSF 100 ng/ml Assaysinclude cell counting (trypan blue) and, typically on days 5, 8 and 11,flow cytometry (CD34, CD90, CD45RA, CD123, CD15, CD33, CD41, CD19).

Bags: 7 ml Vuelife bag (American Fluoroseal, catalog #1PF-0007), 32 ml(#2P-0032) or 72 ml (#2P-0072) were used

Handling of the bags: Use standard precautions for handling human cells.The bags have one (7 ml bags) or two ports that can be used for fillingand drawing samples. The ports have luer locks which allows sample linesand syringes to be connected. The 7 ml bags, once filled with more thanapprox. 4 ml, are under enough pressure to leak when opened, they needto be clamped shut before opening the luer lock. Use syringes to fillthe bags or add more medium, gravity flow is not sufficient. The largerbags can be easily filled by gravity flow, a syringe without plunger canbe connected as a “funnel” when adding medium or cells with a regularpipette. These bags do not need to be clamped prior to opening the lock,they only need to be held upright.

Culturing: The bags are typically cultured in a Sanyo incubator; 37° C.,5% CO₂ and 1-20% 02. The incubators are fully humidified, although thisis not necessary for bag-culturing. The bags are gas-permeable, but notwater permeable. The cell concentration is typically 10⁵ cells/ml(range: 10⁶ cells/ml to 10⁴ cells per ml). The bags may be placed inpetri dishes (15 cm diameter, can hold 7 or 30 ml bags) or in squaredishes (larger bags), for ease of handling and added sterility.

Sampling: Regular cell samples can be drawn from the bags using 1 mlsyringes equipped with a luer lock. Mix the contents of the bag (cellstend to collect in creases, see grey deposits in the picture above).Clamp if necessary (7 ml bag) and remove the stopper from (one of) theports. Attach a 1 ml syringe and invert the bag (syringe down). Removethe clamp, if present. Fill and empty the syringe a few times to mix thecells in the connecting tubes. Empty the syringe (plunger all the waydown) and invert the bag (syringe up). Allow air to move up and seal thehard plastic tube of the sample port (even partially fill it), thenaspire a sample into the tube. The whole of the sample tube containsapprox 0.2 ml. Reclamp if necessary and remove the syringe and replacewith the stopper.

In a 7 ml bag 2 ml of cells in GF containing media, at day 3 or 4additional media was added (2-3 ml) to replenish the culture. In someexperiments, in a 72 ml bag approximately 4×10⁶ cells in 20 ml of medium(cell concentration 2×10⁵ cells per ml). Once cell densities approach10⁶ cells per ml (approximately day 4) the cells are diluted to keep thedensity between 3×10⁵ and 2×10⁶ cells per ml. Media may be added at days4 and 6 (to a total volume of 72 ml) for harvest and freezing at day 8.The cells expanded to approx. 1.5×10⁸ cells in 8 days. A schedule couldbe to start with 3 million cells in 15 ml, add 15 ml of medium on day 4,followed by addition of 40 ml of medium on day 6.

Analysis: Remove a cell sample as described above. Full analysis of thecultures are typically done on days 5, 8 and 12. This includes flowcytometric analysis, plating of 500 cells in 35 mm dishes inmethylcellulose, and May-Grünwald/Giemsa stained cytospins, Cell countsare done on a daily bases using a haemocytometer and trypan blue

Expansion data was obtained from 5 separate donors. FIGS. 11A-11B showsexpansion data in Bags and Flasks from Donor: 1319. FIG. 11A shows totalexpansion, FIG. 11B shows cell density data for the cells (CD34+CD90+)from the donor, cultured in identical medium/GF (Xvivo15 supplementedwith Glutamax, PenStrep, KITL, FLT3L, TPO and IL-3). The cells werecultured at different densities and in different types of bags andflasks. Starting densities between 1×10⁵ and 1×10⁶ cells per ml, both in7 ml AFC bags. Dashed lines indicates cultures that did not receive anymore medium, but were followed to see what the maximal densities werethat could be achieved. Proliferation rates are similar between culturesgrowing in AFC bags and cultures growing in tissue culture flasks. Aproportion of the cells adhered loosely to the plastic of the flasks,this was not the case for the Teflon bags. Phase contrast view of cellsgrowing in flasks and bags Donor: 1319 of cultures at day 8 afterplating show cells in bags tend to gather increases, explaining thegreater apparent density (data not shown).

FIG. 12 is photographs of cells from human MP cultures and treated withgrowth factors. Donor: 1319, cells were cultured in flasks for 8 days(Xvivo15+PenStrep, Glutamax, 100 ng/ml KITL, 100 ng/ml FLT3L, 50 ng/mlTPO and 10 ng/ml IL-3, switched to T25 flasks with different growthfactors (100 ng/ml KITL, 20 ng/ml IL-3 and 300 ng/ml G-CSF). FIG. 12shows cells at different timepoints after transfer (4 to 19 days). Thepresence of granulocytes peaks at day 12, by day 19 only macrophages areseen. Human MP cultures can differentiate into morphologically matureneutrophils and macrophages, as well as megakaryocytes.

Expansion data in bags from Donors 1198 and 1202. Cells from these twodonors were Isolex enriched, sorted for the hpHSC phenotype (CD34⁺CD90⁺)using the Dakocytomation MoFlo and cryopreserved post sorting. The cellswere thawed and plated in 7 ml AFC bags as indicated. Medium: Xvivo15supplemented with Glutamax, PenStrep, KITL, FLT3L, TPO and IL-3. FIG.13A shows total expansion, and FIG. 13B shows cell density data. Opensymbols indicate cells plated at the same time in larger bags (seebelow). No difference is apparent between cells plated at 2×10⁴ cellsper ml and cells plated at 2×10⁵ cells per ml, or between cells platedin 7 ml versus 72 ml AFC bags

Expansion data in bags from Donors 1176, 1198, 1202 and 1207 in 72 mlAFC bags. Cells from these four donors were Isolex enriched, sorted forthe hpHSC phenotype (CD34⁺CD90⁺) using the Dakocytomation MoFlo andcryopreserved post sorting. The cells were thawed and plated in 72 mlAFC bags as indicated. Medium: Xvivo15 supplemented with Glutamax,PenStrep, KITL, FLT3L, TPO and IL-3. FIG. 14A shows total expansion, andFIG. 14B shows cell density data for the cells. Initial plating volume:approx. 4×10⁶ cells in 20 ml, final culture volume 70 ml per bag. Atotal of 15.6×10⁶ cells were plated in the 4 bags, a total of 6.14×10⁸cells were harvested (and cryopreserved) after 8 days. Average expansion40-fold (range 30-fold to 60-fold).

7.4 Example 4. Human Myeloid Progenitors Colony Formation and Responseto G CSF In Vivo

Human myeloid progenitor culture were started with 2×10⁶ purified humanHSC (thawed hpHSC) and cultured in a static AFC bag with serum-freeExVivo-15 with SCF, Flt3L, TPO, IL-3 as described above. At day 5, 8,11, 13 and 15 the MP cells were harvested and plated in triplicates intomethyl cellulose to assess their potential to form colonies in vitro.FIG. 15A shows the plating efficiency (colonies obtained/cells plated)subdivided into the different types of colonies (E: erythroid; M:macrophage; G: granulocyte; GM: mixed granulocyte/macrophage; GEM: mixedmyeloid/erythroid), indicating how many progenitors are present incultures.

FIG. 15B shows the increase of total numbers of CFU (colony formingunits) which is the plating efficiency times total cell count. Althoughthe relative numbers of CFU go down due to the relative strongerincrease in total cell number, there is an increase in CFU over time.

FACS analysis of cultured MP and changes in the stem/progenitorpopulations over time are shown in FIG. 16. Plots shown are pregated onlive and lineage negative cells. The starting population at day 0 isCD34+CD90+(upper right gate) and this population declines over time.Myeloid progenitors are mainly in the CD34+CD90− gate (upper left),additional data indicates the CD34low/− cells make colonies (although toa lower degree) and therefore contribute to the overall progenitor pool(data not shown). The relative number of CD34+CD90− cells against therelative plating efficiency over time was determined (data not show) todetermine the correlation between % CD34+ cells and CFUs. The closecorrelation (data not shown) suggest that CD34 FACS stains can be usedas an indicator for the number of progenitors in cultures.

FIG. 17A-17B show the effect of IL-3 and IL-6, alone and in combinationon human MP cells. FIG. 17A shows cell density and cell count per ml andFIG. 17B show the total cell counts. Human cells were cultured in Xvivocontaining SCF/Flt3L/TPO and the IL-3/IL-6 (10-20 ng/ml) were addedalone or in combination (10-20 ng/ml). The cell counts reveal that IL-3acts as a proliferation factor in the cultures.

FIG. 18A-18C show a result of a colony formation assay of the MP cellscultured with IL-3, IL-6 or both in combination. FIG. 18A (day 5), FIG.18B (day 8), and FIG. 18C (day 11) demonstrates that addition of IL-6increases the numbers of CFU and helps to maintain progenitor potentialof MP cells.

FIG. 19 show the absolute numbers of CFUs in MP cultures with IL-3, IL-6or both in combination. Comparison of the total numbers of CFU from MPcin response to IL-3 and/or IL-6. The proliferate effects of IL-3 and theprogenitor maintaining effects of IL-6, these two cytokines incombination increase the total numbers of colony initiating cells in thecultures.

MPc were culture under standard conditions for 5 (FIG. 20A) or 8 days(FIG. 20B). G-CSF was added 300 ng/ml was added at day 5 or 8 MPc (dayzero on graphs) to the medium and the cell growth monitored over timeand compared to control cultures, which did not receive G-CSF (w/o). Thedata shows that G-CSF can be used to increase cell numbers over longperiod of times when added at later stages to the cultures. It alsoshows that our MP are responsive to G-CSF and are likely to be directprogenitors of granulocytes/neutrophilesm and suggests G-CSF incombination with MPc transplant to increase neutrophils numbers inpatients.

FIG. 21 is a schematic to show responsiveness of human MP cells to G-CSFin vivo. The scheme shows a transplantation experiment of day 8 MPc intoNOD/SCID mice to evaluate their potential to engraft, developmentalpotential and response to G-CSF in vivo.

FIG. 22 is FACS analysis of bone marrow and spleen to look forengraftment of human MPc one week after transplantation and theirresponse to G-CSF. Samples were stained with anti-human CD45 antibody todetect donor cells and shown are two independent samples for each tissuewith/without G-CSF. Bone marrow shows highest degree of reconstitution,which can be increased by injection of G-CSF.

FIG. 23 is FACS phenotype of the human MPc derived cells in NOD/SCIDmice. Plots shown are pregated on live and huCD45+ cells. CD33 is amarker for early myeloid cells and the majority or the human cells areCD33+ indicates that most cells are committed to that lineage. CD14 andCD15 stain more mature myeloid cells and heterogeneous staining showscommitment and maturation into the myeloid lineage. At the same time nohuman B cells (CD19) or T cells (CD3, not shown) were detectable at thattime.

The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,to thereby enable others skilled in the art to best utilize theinvention and various embodiments with various modifications as aresuited to the particular use contemplated.

All patents, patent applications, publications, and references citedherein are expressly incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method of improving impaired hematopoiesis in a human, comprising,administering to the human a composition comprising human allogeneicmyeloid progenitor cells derived from multiple unrelated donors in anamount sufficient to improve hematopoiesis in the human, wherein thereis at least a partial mismatch at a major histocompatability complex(MHC) gene between the donors and the human.
 2. The method of claim 1,wherein the composition has less than 10% hematopoietic stem cells(HSCs).
 3. The method of claim 1, wherein human allogeneic myeloidprogenitor cells are expanded human allogeneic myeloid progenitor cells.4. The method of claim 1, wherein the myeloid progenitor cells comprisecommon myeloid progenitor cells.
 5. The method of claim 1, whereinmyeloid progenitor cells in the composition are at least 25% of totalcells in the composition.
 6. The method of claim 1, wherein the human isundergoing hematopoietic stem cell (HSC) transplantation.
 7. The methodof claim 6, wherein the expanded myeloid progenitor cells areadministered after the HSC transplantation.
 8. The method of claim 6,wherein the expanded myeloid progenitor cells are administeredconcurrently with HSC transplantation.
 9. The method of claim 1, whereinthe human is neutropenic.
 10. The method of claim 1, wherein the humanis suffering from thrombocytopenia.
 11. The method of claim 10, whereinthe expanded myeloid progenitor cells are administered adjunctively witha therapeutic composition for treating complications associated withthrombocytopenia.
 12. The method of claim 11, wherein the therapeuticcomposition comprises a platelet preparation.
 13. The method of claim11, wherein the therapeutic composition comprises EPO.
 14. The method ofclaim 1, wherein the myeloid progenitor cells are expanded from cellsobtained from peripheral blood.
 15. The method of claim 1, wherein themyeloid progenitor cells are expanded from cells obtained from bonemarrow.
 16. The method of claim 1, wherein the myeloid progenitor cellsare expanded from cells obtained from umbilical cord blood or placentalcord blood.
 17. The method of claim 1, wherein the human has beenpreviously treated with or exposed to a myeloablative agent.
 18. Themethod of claim 17, wherein the myeloablative agent is ionizingradiation.
 19. The method of claim 1, wherein prior to theadministering, the human allogeneic myeloid progenitor cells derivedfrom multiple unrelated donors are frozen and subsequently thawed. 20.The method of claim 1, wherein the cells are delivered by intravenousinfusion.d